Keratinocyte growth factor-2 products

ABSTRACT

The present invention concerns variants and chemical derivates of keratinocyte growth factor-2 (KGF-2) protein. Also disclosed are nucleic acid molecules encoding such variants, as well as methods for using such variants and chemical derivates to stimulate epithelial cell proliferation.

This application is a 371 U.S.C. National Phase Filing ofPCT/US97/18607, filed Oct. 15, 1997, which claims the benefit of U.S.Provisional Application No. 60/028,493, filed Oct. 15, 1996; U.S.Provisional Application No. 60/032,781, filed Dec. 6, 1996; and U.S.Provisional Application No. 60/033,046, filed Dec. 10, 1996, all ofwhich are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the use of keratinocyte growth factor-2(KGF-2) protein products to stimulate the proliferation, growth anddifferentiation of a variety of epithelial cells.

BACKGROUND OF THE INVENTION

The complex process of tissue generation and regeneration is mediated bya number of protein factors sometimes referred to as soft tissue growthfactors. These molecules are generally released by one cell type and actto influence proliferation of other cell types (Rubin et al. (1989),Proc. Nat'l. Acad. Sci. USA, 86:802-806). There are also some growthfactors released from cells that themselves have the capacity to respondto such growth factors. Some soft tissue growth factors are secreted byparticular cell types and influence the proliferation, differentiation,and/or maturation of responsive cells in the development ofmulticellular organisms (Finch et al. (1989), Science, 245:752-755). Inaddition to their roles in developing organisms, some soft tissue growthfactors are significant in the continued health and maintenance of moremature systems. For instance, in mammals there are many systems whererapid cell turnover occurs. Such systems include the skin and thegastrointestinal tract, both of which are comprised of epithelial cells.Included within this group of soft tissue growth factors is a proteinfamily of fibroblast growth factors (FGFs).

The fibroblast growth factor (FGF) family is now known to consist of atleast fourteen members, namely FGF-1 to FGF-10 and homologous factorsFHF-1 to FHF-4, which share a relatedness among primary structures:basic fibroblast growth factor, bFGF (Abraham et al. (1986), EMBO J.,5:2523-2528); acidic fibroblast growth factor, aFGF (Jaye et al. (1986),Science, 233:541-545); int-2 gene product, int-2 (Dickson & Peters(1987), Nature, 326:833); hst/kFGF (Delli-Bovi et al. (1987), Cell,50:729-737 and Yoshida et al. (1987), Proc. Natl. Acad. Sci. USA,84:7305-7309); FGF-5 (Zhan et al. (1988), Mol. Cell. Biol.,8:3487-3495); FGF-6 (Marics et al. (1989), Oncogene, 4:335-340);keratinocyte growth factor, KGF (Finch et al. (1989), Science,24:752-755); hisactophilin (Habazzettl et al. (1992), Nature,359:855-858); FGF-9 (Miyamoto et al. (1993), Mol. Cell Biol.,13(7):4251-4259); and fibroblast growth factor-10, also known askeratinocyte growth factor-2, KGF-2 (PCT patent application WO96/25422), the disclosures of which are hereby incorporated byreference. More recently, four homologous factors (or “FHFs”) wereidentified from the human retina by a combination of random cDNAsequencing, searches of existing sequence databases and homology-basedpolymerase chain reactions (Smallwood et al. (1996), Proc. Natl. Acad.Sci. USA, 93:9850-9857). It has been proposed that FHF-1, FHF-2, FHF-3and FHF-4 should be designated as FGF-11, FGF-12, FGF-13 and FGF-14,respectively, in accordance with the recommendation of the NomenclatureCommittee (Coulier et al. (1997), Journal of Molecular Evolution,44:43-56, the disclosure of which is hereby incorporated by reference).

WO 96/25422 describes the cloning, expression and purification offull-length (with signal sequence, residues Met¹ to Thr³⁶ of SEQ IDNO:2) and mature (without signal sequence, residues Cys³⁷ to Ser²⁰⁸ ofSEQ ID NO:2) KGF-2 in a bacterial expression system (e.g., E. coli) andeukaryotic expression systems (e.g., baculovirus and COS cells). Thisreference further teaches that KGF-2 might be useful to stimulate cellgrowth and proliferation for new blood vessel growth or angiogenesis,the prevention of hair loss, the healing of dermal wounds and thedifferentiation of muscle cells, nervous tissue, prostate cells and lungcells.

Much remains to be learned regarding KGF-2, including modificationswhich can be made thereto to generate variant(s) and derivatives whichretain some or all of the biological activity of KGF-2. Generally, theeffects of any specific amino acid change or chemical derivatizationupon biological activity of a protein will vary depending upon a numberof factors, including whether or not modifications affect thethree-dimensional structure or the receptor binding region of theprotein. As neither the three-dimensional structure nor the receptorbinding region of KGF-2 has been published, the knowledge within the artdoes not permit generalization about the effects of specific amino acidmodifications or chemical derivatization to KGF-2.

It is the object of this invention to provide variants and derivativesof KGF-2 that retain some or all of the biological activity of KGF-2.

SUMMARY OF THE INVENTION

The present invention is directed to KGF-2 protein product(s), asdefined below. These KGF-2 protein product(s) have general applicabilityand may retain some or all of the biological activity of KGF-2.

In one aspect, a variant(s) of KGF-2 is produced by recombinant geneticengineering techniques. In an alternative embodiment, a variant(s) ofKGF-2 is synthesized by chemical techniques, or a combination of therecombinant and chemical techniques A variant(s) of KGF-2 may be made inglycosylated or non-glycosylated form.

Yet another aspect of the present invention includes the variouspolynucleotides encoding a variant(s) of KGF-2. Each such nucleic acidsequence may be used in the expression of a variant(s) of KGF-2 in aeukaryotic or prokaryotic host cell. The polynucleotides may also beused in cell therapy or gene therapy applications.

A further aspect of the present invention involves vectors containingthe polynucleotides encoding a variant(s) of KGF-2 operatively linked toamplification and/or expression control sequences.

A still further aspect of the present invention pertains to bothprokaryotic and eukaryotic host cells containing recombinantpolynucleotides encoding variant(s) of KGF-2.

In another aspect, the present invention further includes therecombinant production of a variant(s) of KGF-2 wherein recombinant hostcells are grown in a suitable nutrient medium and wherein a variant(s)of KGF-2 expressed by the cells is, optionally, isolated from the hostcells and/or the nutrient medium.

A still further aspect of the present invention includes KGF-2protein(s), as defined below, attached to a water soluble polymer. Forexample, a variant(s) of KGF-2 may be conjugated to one or morepolyethylene glycol molecules in order to improve pharmacokineticperformance by increasing the molecule's apparent molecular weight.

Another aspect of the present invention includes pharmaceuticalcompositions containing a variant(s) of KGF-2, or chemical derivative(s)of KGF-2 protein(s). Typically, a variant(s) of KGF-2 may be formulatedin association with pharmaceutically acceptable vehicles. A variety ofother formulation materials may be used to facilitate the manufacture,storage, handling, delivery and/or efficacy of a variant(s) of KGF-2.

Yet another aspect relates to methods of modulating the growth anddifferentiation of epithelial cells. Specifically, a patient in need ofstimulation (including cytoprotection, proliferation and/ordifferentiation) of epithelial cells will be administered atherapeutically-effective or prophylactically-effective amount of avariant(s) of KGF-2 and/or a chemical derivative of KGF-2 protein(s).

Additional aspects and advantages of the invention will be apparent tothose skilled in the art upon consideration of the followingdescription, which details the practice of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Numerous aspects and advantages of the present invention will becomeapparent upon review of the Figures, wherein:

FIG. 1 depicts a cDNA sequence (SEQ ID NO:1) encoding full-length,recombinant human KGF-2. Also depicted is the amino acid sequence (SEQID NO:2) of full-length, recombinant human KGF-2. The initial 36 aminoacid residues (Met¹ to Thr³⁶) represent the putative leader sequence offull-length KGF-2, and residues Cys³⁷ to Ser²⁰⁸ of SEQ ID NO:2 representmature KGF-2. The full-length and mature forms are collectively termed“KGF-2”.

FIG. 2 depicts a cDNA sequence (SEQ ID NO:3) encoding dN29 hFGF10. Alsodepicted is the amino acid sequence (SEQ ID NO:4) of dN29 hFGF10.

FIG. 3 depicts a cDNA sequence (SEQ ID NO:5) encoding dN20 hFGF10. Alsodepicted is the amino acid sequence (SEQ ID NO:6) of dN20 hFGF10.

FIG. 4 depicts a cDNA sequence (SEQ ID NO:7) encoding hFGF10 R149Q. Alsodepicted is the amino acid sequence (SEQ ID NO:8) of hFGF10 R149Q.

DETAILED DESCRIPTION OF THE INVENTION

KGF-2 Protein(s)

In accordance with the terms of this invention, by the term “KGF-2protein(s)” is meant the protein defined by amino acids Cys³⁷ to Ser²⁰⁸of SEQ ID NO:2 (mature KGF-2) and variant proteins thereof. The term“KGF-2 protein(s)” thus includes a protein in which one or more aminoacid residues have been deleted from (“deletion variant(s)”), insertedinto (“addition variant(s)”), and/or substituted for (“substitutionvariant(s)”) residues within the amino acid sequence of SEQ ID NO:2 andwhich retains biological activity. Thus, while the descriptions below ofprotein modifications refer to mature KGF-2, it does not precludeadditional modifications thereto.

The term “biological activity” as used herein means that a KGF-2protein(s) possesses some but not necessarily all the same properties of(and not necessarily to the same degree as) mature KGF-2. The selectionof the particular properties of interest depends upon the desired use ofthe desired KGF-2 protein(s).

It will be appreciated by those skilled in the art that manycombinations of deletions, insertions, and substitutions can be made,provided that the final protein is biologically active. There are twoprincipal variables in the construction of amino acid sequencevariant(s): the location of the mutation site and the nature of themutation. In designing a variant(s), the location of the mutation siteand the nature of the mutation will depend on the biochemicalcharacteristic(s) to be modified. Mutation sites can be modifiedindividually or in series, e.g., by (1) deleting the target amino acidresidue, (2) inserting amino acid residues adjacent to the located siteor (3) substituting first with conservative amino acid choices and,depending upon the results achieved, then with more radical selections.

Amino acid sequence deletions generally range from about 40 amino acidresidues, from about 30 amino acids, from about 20 amino acids, andtypically from about 1 to 10 residues. Deletions within the amino acidsequence of mature KGF-2 may be made, for example, in regions of lowhomology with the sequences of other members of the FGF family.Deletions within the amino acid sequence of mature KGF-2 in areas ofsubstantial homology with the sequences of other members of the FGFfamily will be more likely to significantly modify the biologicalactivity. The number of total deletions and/or consecutive deletionspreferably will be selected so as to preserve the tertiary structure ofmature KGF-2 (amino acids Cys³⁷ to Ser²⁰⁸ of SEQ ID NO:2) in theaffected domain, e.g., cysteine crosslinking.

Amino acid sequence additions may include amino- and/orcarboxyl-terminal fusions ranging in length from one residue to onehundred or more residues, as well as internal intrasequence insertionsof single or multiple amino acid residues. Internal additions may rangepreferably from about 1 to 10 amino acid residues, more preferably fromabout 1 to 5 amino acid residues, and most preferably from about 1 to 3amino acid residues. Additions within the amino acid sequence of matureKGF-2 may be made in regions of low homology with the sequences of othermembers of the FGF family. Additions within the amino acid sequence ofmature KGF-2 in areas of substantial homology with the sequences ofother members of the FGF family will be more likely to significantlymodify the biological activity. Insertions or additions preferablyinclude amino acid sequences derived from the sequences of other FGFfamily members.

An amino-terminus addition is contemplated to include the addition of amethionine (for example, as an artifact of the direct expression inbacterial recombinant cell culture) or an amino acid residue or sequenceof mature KGF-2. A further example of an N-terminal addition includesthe fusion of a signal sequence to the N-terminus of mature KGF-2 inorder to facilitate the secretion of protein from recombinant hostcells. Such signal sequences generally will be obtained from and thus behomologous to the intended host cell species. Included within the scopeof this invention is the native signal sequence, for example, the nativesignal sequence of the protein defined by amino acids Met¹ to Thr³⁶ ofSEQ ID NO:2 or a heterologous signal sequence. A heterologous signalsequence selected should be one that is recognized and processed (i.e.,cleaved by a signal peptidase) by the host cell. For prokaryotic hostcells that do not recognize and process the native signal sequence, thesignal sequence may be substituted by a prokaryotic signal sequenceselected, for example, from the group of the alkaline phosphatase,penicillinase or heat-stable enterotoxin II leaders. For yeastsecretion, the signal sequence may be selected, for example, from thegroup of the yeast invertase, alpha factor or acid phosphatase leadersequences. In mammalian cell expression specifically, signal sequencesof full-length KGF-2 or of other FGF family members (e.g., KGF) may besuitable.

An example of a carboxy-terminus addition includes chimeric proteinscomprising the fusion of KGF-2 with all or part of the constant domainof the heavy or light chain of,human immunoglobulin. Such chimericpolypeptides are preferred wherein the immunoglobulin portion comprisesall the domains except the first domain of the constant region of theheavy chain of a human immunoglobulin such as IgG, IgA, IgM or IgE,especially IgG, e.g., IgG1 or IgG3. A skilled artisan will appreciatethat any amino acid of the immunoglobulin portion can be deleted orsubstituted by one or more amino acids, or one or more amino acids canbe added as long as the KGF-2 portion still stimulates epithelial cellsand the immunoglobulin portion shows one or more of its characteristicproperties.

Another group of variant(s) is amino acid substitution variant(s) of theamino acid sequence of mature KGF-2. These variant(s) have at least oneamino acid residue in the sequence of Cys³⁷ to Ser²⁰⁸ of SEQ ID NO:2removed and a different residue inserted in its place. Substitutionvariant(s) include allelic variant(s), which are characterized bynaturally-occurring nucleotide sequence changes in the speciespopulation that may or may not result in an amino acid change. Oneskilled in the art can use any information known about the binding oractive site of the polypeptide in the selection of possible mutationsites.

One method for identifying amino acid residues or regions formutagenesis is called “alanine scanning mutagenesis”, as described byCunningham and Wells (1989), Science, 244:1081-1085, the disclosure ofwhich is hereby incorporated by reference. In this method, an amino acidresidue or group of target residues is identified (e.g., chargedresidues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutralor negatively-charged amino acid (most preferably alanine orpolyalanine) to effect the interaction of the amino acids with thesurrounding aqueous environment in or outside the cell. Those domainsdemonstrating functional sensitivity to the substitutions are thenrefined by introducing additional or alternate residues at the sites ofsubstitution. Thus, the site for introducing an amino acid sequencemodification is predetermined and, to optimize the performance of amutation at a given site, alanine scanning or random mutagenesis may beconducted and the variant(s) may be screened for the optimal combinationof desired activity and degree of activity.

The sites of greatest interest for substitutional mutagenesis includesites in which particular residues within amino acids Cys³⁷ to Ser²⁰⁸ ofSEQ ID NO:2 are substantially different from various species or otherFGF family members in terms of side-chain bulk, charge, and/orhydrophobicity. Other sites of interest include those in whichparticular residues within amino acids Cys³⁷ to Ser²⁰⁸ of SEQ ID NO:2,are identical among various species or other FGF family members. Suchpositions are generally important for the biological activity of aprotein. Accordingly, a skilled artisan would appreciate that initiallythese sites should be modified by substitution in a relativelyconservative manner.

Such conservative substitutions are shown in Table 1 under the headingof “Preferred Substitutions”. If such substitutions result in a changein biological activity, then more substantial changes (ExemplarySubstitutions) may be introduced and/or other additions/deletions may bemade and the resulting products screened.

TABLE 1 Amino Acid Substitutions Original Preferred Exemplary ResidueSubstitutions Substitutions Ala (A) Val Val; Leu; Ile Arg (R) Lys Lys;Gln; Asn Asn (N) Gln Gln; His; Lys; Arg Asp (D) Glu Glu Cys (C) Ser SerGln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Pro His (H) Arg Asn; Gln;Lys; Arg Ile (I) Leu Leu; Val; Met; Ala; Phe; norleucine Leu (L) Ilenorleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met (M)Leu Leu; Phe; Ile Phe (F) Leu Leu; Val; Ile; Ala Pro (P) Gly Gly Ser (S)Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Phe Trp; Phe; Thr; SerVal (V) Leu Ile; Leu; Met; Phe; Ala; norleucine

In making such changes of an equivalent nature, the hydropathic index ofamino acids may be considered. The importance of the hydropathic aminoacid index in conferring interactive biological function on a protein isgenerally understood in the art (Kyte and Doolittle (1982), J. Mol.Biol., 157:105-131, the disclosure of which is incorporated herein byreference). It is known that certain amino acids may be substituted forother amino acids having a similar hydropathic index or score and stillretain a similar biological activity.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biological functional equivalent protein orpeptide thereby created is intended for use in immunologicalembodiments, as in the present case. U.S. Pat. No. 4,554,101, thedisclosure of which is incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsimmunogenicity and antigenicity, i.e., with a biological property of theprotein.

U.S. Pat. No. 4,554,101 also teaches the identification and preparationof epitopes from primary amino acid sequences on the basis ofhydrophilicity. Through the methods disclosed in U.S. Pat. No. 4,554,101a skilled artisan would be able to identify epitopes, for example,within the amino acid sequence of KGF-2. These regions are also referredto as “epitopic core regions”. Numerous scientific publications havebeen devoted to the prediction of secondary structure, and to theidentification of epitopes, from analyses of amino acid sequences (Chouand Fasman (1974), Biochemistry, 13(2):222-245; Chou and Fasman (1974),Biochemistry, 13(2):211-222; Chou and Fasman (1978), Adv. Enzymol.Relat. Areas Mol. Biol., 47:45-148; Chou and Fasman (1978), Ann. Rev.Biochem., 47:251-276 and Chou and Fasman (1979), Biophys. J.,26:367-384, the disclosures of which are incorporated herein byreference). Moreover, computer programs are currently available toassist with predicting antigenic portions and epitopic core regions ofproteins. Examples include those programs based upon the Jameson-Wolfanalysis (Jameson and Wolf (1988), Comput. Appl. Biosci., 4(1):181-186and Wolf et al. (1988), Comput. Appl. Biosci., 4(1):187-191, thedisclosures of which are incorporated herein by reference); the programPepPlot® (Brutlag et al. (1990), CABS, 6:237-245 and Weinberger et al.(1985), Science, 228:740-742, the disclosures of which are incorporatedherein by reference); and other new programs for protein tertiarystructure prediction (Fetrow and Bryant (1993), BIOTECHNOLOGY,11:479-483, the disclosure of which is incorporated herein byreference).

In contrast, substantial modifications in the functional and/or chemicalcharacteristics of the amino acids Cys³⁷ to Ser²⁰⁸ of SEQ ID NO:2 may beaccomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the relative charge or hydrophobicity of theprotein at the target site or (c) the bulk of the side chain.Naturally-occurring residues are divided into groups based on commonside chain properties

1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr;

3) acidic: Asp, Glu;

4) basic: Asn, Gln, His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions may involve the exchange of a member ofone of these groups for another. Such substituted residues may beintroduced into regions of amino acids Cys³⁷ to Ser²⁰⁸ of SEQ ID NO:2that, for example, are homologous with regions of other FGF familymembers or into the non-homologous regions of the protein.

In a specific embodiment, a variant polypeptide will preferably besubstantially homologous to amino acids Cys³⁷ to Ser²⁰⁸ of SEQ ID NO:2.The term “substantially homologous”, as used herein, means having adegree of homology (i.e., identity of amino acid residues) to aminoacids Cys³⁷ to Ser²⁰⁸ of SEQ ID NO:2 in excess of eighty percent (80%);preferably, in excess of ninety percent (90%); more preferably, inexcess of ninety-five percent (95%); and most preferably, in excess ofninety-nine percent (99%). The percentage of homology as describedherein is calculated as the percentage of amino acid residues found inthe smaller of the two sequences which align with identical amino acidresidues in the sequence being compared when four gaps in a length of100 amino acids may be introduced to assist in that alignment, as setforth by Dayhoff (1972), in Atlas of Protein Sequence and Structure,5:124, National Biochemical Research Foundation, Washington, D.C., thedisclosure of which is hereby incorporated by reference. Also includedas substantially homologous are variant(s) of the amino acids Cys³⁷ toSer²⁰⁸ of SEQ ID NO:2 which may be isolated by virtue ofcross-reactivity with antibodies to amino acids Cys³⁷ to Ser²⁰⁸ of SEQID NO:2, or whose genes may be isolated through hybridization with theDNA of SEQ ID NO:1 or with segments thereof.

A first class of variant(s) is a group of deletion variants of Cys³⁷ toSer²⁰⁸ of SEQ ID NO:2. These variants include R₁-[Asn⁷¹-Pro²⁰³]-R₂—COOHproteins, and further include an amino acid sequence comprisingNH₂-[His⁷²-Ser²⁰⁸]-COOH (also referred to as ΔN35 KGF-2),NH₂-[Leu⁷³-Ser²⁰⁸]-COOH (also referred to as ΔN36 KGF-2),NH₂-[Gln⁷⁴-Ser²⁰⁸]-COOH (also referred to as ΔN37 KGF-2),NH₂-[Gly⁷⁵-Ser²⁰⁸]-COOH (also referred to as ΔN38 KGF-2),NH₂-[Asp⁷⁶-Ser²⁰⁸]-COOH (also referred to as ΔN39 KGF-2),NH₂-[Val⁷⁷-Ser²⁰⁸]-COOH (also referred to as ΔN40 KGF-2) andNH₂-[Arg⁷⁸-Ser²⁰⁸]-COOH (also referred to as ΔN41 KGF-2), in which eachmay be N-terminally methionylated or non-methionylated, provided howeverthat Cys³⁷ to Ser²⁰⁸ of SEQ ID NO:2 is excluded.

By “R₁-[Asn⁷¹-Pro²⁰³]-R₂—COOH” is meant a group of deletion variant(s),wherein [Asn⁷¹-Pro203] represents residues 71 through 203 of SEQ IDNO:2; wherein R₁ represents a methionylated or nonmethionylated aminegroup of Asn⁷¹ or of amino-terminus amino acid residue(s) selected fromthe group:

Tyr

Ser-Tyr

Arg-Ser-Tyr

Val-Arg-Ser-Tyr (SEQ ID NO:9),

His-Val-Arg-Ser-Tyr (SEQ ID NO:10),

Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:11),

Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:12),

Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:13),

Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:14),

Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:15),

Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:16),

Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:17),

Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:18),

Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ ID NO:19),

Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ IDNO:20),

Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ IDNO:21),

Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr (SEQ IDNO:22),

Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr(SEQ ID NO:23),

Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr(SEQ ID NO:24),

Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr(SEQ ID NO:25),

Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr(SEQ ID NO:26),

Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr(SEQ ID NO:27),

Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr(SEQ ID NO:28),

Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr(SEQ ID NO:29),

Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr(SEQ ID NO:30),

Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr(SEQ ID NO:31),

Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr(SEQ ID NO:32),

Asp-Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr(SEQ ID NO:33),

Gln-Asp-Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr(SEQ ID NO:34),

Gly-Gln-Asp-Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr(SEQ ID NO:35),

Leu-Gly-Gln-Asp-Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr(SEQ ID NO:36),

Ala-Leu-Gly-Gln-Asp-Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr(SEQ ID NO:37),

Gln-Ala-Leu-Gly-Gln-Asp-Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr(SEQ ED NO:38), or

Cys-Gln-Ala-Leu-Gly-Gln-Asp-Met-Val-Ser-Pro-Glu-Ala-Thr-Asn-Ser-Ser-Ser-Ser-Ser-Phe-Ser-Ser-Pro-Ser-Ser-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr(SEQ ID NO:39),

and, wherein R₂ represents a carboxy group of Pro²⁰³ or ofcarboxy-terminal amino acid residues of:

Met

Met-Val-Val

Met-Val-Val-His (SEQ ID NO:40), or

Met-Val-Val-His-Ser (SEQ ID NO:41),

provided however, that R₁ and R₂ are not selected so as to reconstructCys³⁷ to Ser²⁰⁸ of SEQ ID NO:2.

Exemplary substitutions of KGF-2 and of variant(s) of KGF-2(particularly R₁-[Asn⁷¹-Pro²⁰³]-R₂—COOH proteins, and more particularlyΔN36 KGF-2, ΔN35 KGF-2, ΔN34 KGF-2, ΔN33 KGF-2, ΔN32 KGF-2, ΔN31 KGF-2,ΔN30 KGF-2, ΔN29 KGF-2, ΔN28 KGF-2, ΔN27 KGF-2 and ΔN26 KGF-2, eithermethionylated or nonmethionylated) are set forth in the following table:

Preferred variant(s) within this class include the following molecules:ΔN36 KGF-2; ΔN35 KGF2; NH₂-[Asn⁷¹-Ser²⁰⁸]-COOH (also referred to as ΔN34KGF-2); NH₂-Tyr-[Asn⁷¹-Ser²⁰⁸]-COOH (also referred to as ΔN33 KGF-2);NH₂-Ser-Tyr-[Asn⁷¹-Ser²⁰⁸]-COOH (also referred to as ΔN32 KGF-2);NH₂-Arg-Ser-Tyr-[Asn⁷¹-Ser²⁰⁸]-COOH (also referred to as ΔN31 KGF-2);NH₂-Val-Arg-Ser-Tyr-[Asn⁷¹-Ser²⁰⁸]-COOH (also referred to as ΔN30KGF-2); NH₂-His-Val-Arg-Ser-Tyr-[Asn⁷¹-Ser²⁰⁸]-COOH (also referred to asΔN29 KGF-2); NH₂-Arg-His-Val-Arg-Ser-Tyr-[Asn⁷¹-Ser²⁰⁸]-COOH (alsoreferred to as ΔN28 KGF-2);NH₂-Gly-Arg-His-Val-Arg-Ser-Tyr-[Asn⁷¹-Ser²⁰⁸]-COOH (also referred to asΔN27 KGF-2); and NH₂-Ala-Gly-Arg-His-Val-Arg-Ser-Tyr-[Asn⁷¹-Ser²⁰⁸]-COOH(also referred to as ΔN26 KGF-2), either methionylated ornonmethionylated.

A second class of variant(s) is a group of substitution, deletion oraddition variant(s) of KGF-2 and/or of the first class of KGF-2variant(s), described above, having a region corresponding to Asn¹⁶⁸ toMet¹⁷⁶ of SEQ ID NO:2 wherein at least one amino acid residue within theregion corresponding to Asn¹⁶⁸ to Met¹⁷⁶ of SEQ ID NO:2 is deleted orsubstituted with a non-native amino acid, or a non-native amino acid isadded within the region corresponding to Asn¹⁶⁸ to Met¹⁷⁶ of SEQ IDNO:2; this region is unique among the FGF family and contains residues(Trp¹⁶⁹ and His¹⁷¹) which may predominantly confer binding specificityand residues (Gly¹⁷³ and Met¹⁷⁶) which may predominantly stabilize thestructure to the region. In a specific embodiment, the regioncorresponding to Asn¹⁶⁸ to Met¹⁷⁶ is deleted or replaced with thefollowing sequence: NH₂-Ala-Lys-Trp-Thr-His-Asn-Gly-Gly-Glu-Met-COOH,which is the sequence of a putative receptor binding region of KGF.

A third class of variant(s) is a group of deletion or substitutionvariant(s) of KGF-2 and/or of the first class of KGF-2 variant(s) and/orof the second class of KGF-2 variant(s), described above, having aregion corresponding to Phe⁸⁵ to Ser¹⁹⁸ of SEQ ID NO:2 wherein at leastone neutral or positively charged amino acid residue within the regioncorresponding to Phe⁸⁵ to Ser¹⁹⁸ of SEQ ID NO:2 is deleted orsubstituted with a neutral residue or negatively charged residueselected to effect a charge-change protein with a reduced positivecharge. Preferred residues for modification are residues correspondingto Phe⁸⁵, Thr⁸⁶, Asn¹⁵⁹, Gly¹⁸², Arg¹⁸⁷, Asn¹⁹⁶, Thr¹⁹⁷, Ser¹⁹⁸ of SEQID NO:2, with residues Thr⁸⁶, Gly¹⁸², Arg¹⁸⁷ and Asn¹⁹⁶ being morepreferred. Preferred amino acids for substitution include alanine,glutamic acid, aspartic acid, glutamine, asparagine, glycine, valine,leucine, isoleucine, serine and threonine; with alanine, glutamic acid,glutamine, aspartic acid and asparagine being more preferred; and withalanine being most preferred.

A fourth class of variant(s) is a group of substitution variant(s) ofKGF-2 and/or of the first class of KGF-2 variant(s) and/or of the secondclass of KGF-2 variant(s) and/or of the third class of KGF-2 variant(s),described above, having a region corresponding to a putative surfaceloop-forming region, Asn¹⁶⁰ to Thr¹⁶⁴ of SEQ ID NO:2, wherein at leastone amino acid having a higher loop forming potential is substituted forat least one amino acid having a lower loop forming potential within theregion corresponding to Asn¹⁶⁰ to Thr¹⁶⁴ of SEQ ID NO:2. The non-nativeamino acid is selected for its higher loop-forming potential in order tostabilize this area of the protein. Amino acids having relatively highloop-forming potential include glycine, proline, tyrosine, asparticacid, asparagine, and serine. Leszcynski et al, Science, 234, 849-855(1986) (relative values of loop-forming potential assigned on the basisof frequency of appearance in loop structures of naturally occurringmolecules). Preferably, a different amino acid having higherloop-forming potential replaces a threonine residue corresponding toThr¹⁶⁴ of SEQ ID NO:2 in the loop-forming sequence.

A fifth class of variant(s) is a group of substitution variant(s) ofKGF-2 and/or of the first class of KGF-2 variant(s) and/or of the secondclass of KGF-2 variant(s) and/or of the third class of KGF-2 variant(s)and/or of the fourth class of KGF-2 variant(s), described above, havingamino acid residues corresponding to Cys³⁷, Cys¹⁰⁶ or Cys¹⁵⁰ of SEQ IDNO:2 wherein at least one naturally-occurring cysteine residue at aposition corresponding to position 37, 106 or 150 of SEQ ID NO:2 isdeleted or substituted with a non-native amino acid residues (e.g., Ala,Leu, or Ser).

A sixth class of variant(s) is a group of substitution or deletionvariant(s) of KGF-2 and/or of the first class of KGF-2 variant(s) and/orof the second class of KGF-2 variant(s) and/or of the third class ofKGF-2 variant(s) and/or of the fourth class of KGF-2 variant(s) and/orof the fifth class of KGF-2 variant(s), described above, having at leastone N-linked or O-linked glycosylation site corresponding to an N-linkedor O-linked glycosylation site within Cys³⁷ to Ser²⁰⁸ of SEQ ID NO:2.Such variant(s) have at least one amino acid within the N-linked orO-linked glycosylation site within the region corresponding to anN-linked or O-linked glycosylation site within Cys³⁷ to Ser²⁰⁸ of SEQ IDNO:2 deleted or substituted with a non-native amino acid, to modify theN-linked or O-linked glycosylation site and generate a protein withaltered glycosylation. An asparagine-linked glycosylation recognitionsite comprises a tripeptide sequence which is specifically recognized byappropriate cellular glycosylation enzymes. These tripeptide sequencesare either Asn-Xaa-Thr or Asn-Xaa-Ser, where Xaa can be any amino acidother than Pro. Proven or predicted asparagine residues exist atpositions 51 and 196 of amino acids Cys³⁷ to Ser²⁰⁸ of SEQ ID NO:2. Avariety of amino acid substitutions or deletions may be made to modifyN-linked or O-linked glycosylation sites.

A seventh class of variant(s) is a group of addition variant(s) of KGF-2and/or of the first class of KGF-2 variant(s) and/or of the second classof KGF-2 variant(s) and/or of the third class of KGF-2 variant(s) and/orof the fourth class of KGF-2 variant(s) and/or of the fifth class ofKGF-2 variant(s) and/or of the sixth class of KGF-2 variant(s) describedabove, wherein putative cleavage sites Asn¹²⁸-Gly¹²⁹ and/orAsn¹⁴¹-Gly¹⁴² are modified by a substitution of an amino acid (e.g., Glnor Ser) for the Asn¹²⁸ and/or Asn¹⁴¹.

A eighth class of variant(s) is a group of addition variant(s) of KGF-2and/or of the first class of KGF-2 variant(s) and/or of the second classof KGF-2 variant(s) and/or of the third class of KGF-2 variant(s) and/orof the fourth class of KGF-2 variant(s) and/or of the fifth class ofKGF-2 variant(s) and/or of the sixth class of KGF-2 variant(s) and/or ofthe seventh class of KGF-2 variant(s), described above, wherein fused tothe C-terminus of one of the aforementioned proteins is animmunoglobulin portion comprising at least one domain of the constantregion of the heavy chain of human immunoglobulin (however, generallyexcluding the first domain) such as IgG, IgA, IgM or IgE, especiallyIgG, e.g., IgG1 or IgG3.

Exemplary substitutions of KGF-2 and of variant(s) of KGF-2(particularly R₁-[Asn⁷¹-Pro²⁰³]-COOH proteins, and more particularlyΔN36 KGF-2, ΔN35 KGF-2, ΔN34 KGF-2, ΔN33 KGF-2, ΔN32 KGF-2, ΔN31 KGF-2,ΔN30 KGF-2, ΔN29 KGF-2, ΔN28 KGF-2, ΔN27 KGF-2 and ΔN26 KGF-2, eithermethionylated or nonmethionylated) are set forth in the following table:

TABLE 2 Original residue Preferred Substitution Asn⁷¹ Arg, Asp, Glu, LysLeu⁸² Gly Phe⁸⁵ Arg, Tyr Thr⁸⁶ Ala, Asp, Glu, Gly, Ser Glu⁹³ Asp Lys¹⁰²Gln, Glu Lys¹⁰³ Glu, Gln Glu¹⁰⁴ Met Cys¹⁰⁶ Ser, Ala, Met, Asn Pro¹⁰⁷Ala, Asn, Gly Tyr¹⁰⁸ Ala, Phe, Ser Leu¹¹¹ Ala, Met, Ser Thr¹¹⁴ Ala, Arg,Lys, Ser Val¹²³ Ile, Leu Asn¹²⁷ Asp, Glu, Lys Tyr¹³⁰ Phe Gly¹⁴² Ala, SerSer¹⁴³ Ala, Glu, Lys Phe¹⁴⁶ Tyr, Ser, Met Leu¹⁵² Ala, Ile, Met, PheAsn¹⁵⁹ Ala, Asp, Gln Gly, Glu, Ile, Lys, Met Gly¹⁶⁰ Ala, His, Ser, TyrPhe¹⁶⁷ Ala, Ser, Tyr Gln¹⁷⁰ Arg, Glu, Ser, Thr Arg¹⁷⁴ Gly, Ala, SerTyr¹⁷⁷ Phe, Leu Gly¹⁸² Ala, Asp, Glu, Ser Arg¹⁸⁷ Ala, Glu, Gly, SerArg¹⁸⁸ Gln Lys¹⁹⁵ Glu, Gln Asn¹⁹⁶ Ala, Arg, Asp, Gln, Glu, Gly, LysThr¹⁹⁷ Ala, Arg, Asp, Lys, Glu, Gly Ser¹⁹⁸ Ala, Asp, Glu, Gly, ThrVal²⁰⁶ Ala, Ile, Leu, Val His²⁰⁷ Leu, Ser, Thr, Tyr

It will be appreciated by those skilled in the art that manycombinations of deletions, insertions and substitutions can be made,provided that the final KGF-2 protein(s) are biologically active. Avariant(s) of KGF-2 may be rapidly screened to assess its physicalproperties. For example, the level of biological activity (e.g.,receptor binding and/or affinity, mitogenic, cell proliferative and/orin vivo activity) may be tested using a variety of assays. One suchassay includes a mitogenic assay to test the ability of a protein tostimulate DNA synthesis (Rubin et al. (1989), supra, the disclosure ofwhich is hereby incorporated by reference). Another such assay includesa cell proliferative assay to test the ability of a protein to stimulatecell proliferation (Falco et al. (1988), Oncogene, 2:573-578, thedisclosure of which is hereby incorporated by reference).

Polypeptide Derivatives

Chemically-modified derivatives of KGF-2 protein(s) in which thepolypeptide is linked to a polymer in order to modify properties(referred herein as “derivatives”), are included within the scope of thepresent invention. Chemically-modified derivatives of KGF-2 protein(s)may be prepared by one skilled in the art given the disclosures herein.Conjugates may be prepared using glycosylated, non-glycosylated orde-glycosylated KGF-2 protein(s). Typically, non-glycosylated KGF-2protein(s) will be used.

Suitable chemical moieties for derivatization include water solublepolymers. Water soluble polymers are desirable because the protein towhich each is attached will not precipitate in an aqueous environment,such as a physiological environment. Preferably, the polymer will bepharmaceutically acceptable for the preparation of a therapeutic productor composition. One skilled in the art will be able to select thedesired polymer based on such considerations as whether thepolymer/protein conjugate will be used therapeutically and, if so, thetherapeutic profile (e.g., the duration of sustained release; resistanceto proteolysis, the effects, if any, on dosage, biological activity; theease in handling; the degree or lack of antigenicity and other knowneffects of a water soluble polymer on a therapeutic protein).

Suitable, clinically acceptable, water soluble polymers include, but arenot limited to, polyethylene glycol (PEG), polyethylene glycolpropionaldehyde, copolymers of ethylene glycol/propylene glycol,monomethoxy-polyethylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, (PVA), polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, poly (β-aminoacids) (either homopolymers or random copolymers), poly(n-vinylpyrrolidone)polyethylene glycol, polypropylene glycol homopolymers (PPG)and other polyalkylene oxides, polypropylene oxide/ethylene oxidecopolymers, polyoxyethylated polyols (POG) (e.g., glycerol) and otherpolyoxyethylated polyols, polyoxyethylated sorbitol, or polyoxyethylatedglucose, colonic acids or other carbohydrate polymers, Ficoll or dextranand mixtures thereof. As used herein, polyethylene glycol is meant toencompass any of the forms that have been used to derivatize otherproteins, such as mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water.

The water soluble polymers each may be of any molecular weight and maybe branched or unbranched. Generally, the higher the molecular weight orthe more branches, the higher the polymer:protein ratio. The watersoluble polymers each typically have an average molecular weight ofbetween about 2 kDa to about 100 kDa (the term “about” indicating thatin preparations of a water soluble polymer, some molecules will weighmore, some less, than the stated molecular weight). The averagemolecular weight of each water soluble polymer preferably is betweenabout 5 kDa and about 40 kDa, more preferably between about 10 kDa andabout 35 kDa and most preferably between about 15 kDa and about 30 kDa.

There are a number of attachment methods available to those skilled inthe art, including acylation reactions or alkylation reactions(preferably to generate an N-terminal chemically modified protein) witha reactive water soluble molecule. See, for example, EP 0 401 384, thedisclosure of which is hereby incorporated by reference; see also, Maliket al. (1992), Exp. Hematol., 20:1028-1035; Francis (1992), Focus onGrowth Factors, 3(2):4-10, published by Mediscript, Mountain Court,Friern Barnet Lane, London N20 OLD, UK; EP 0 154 316; EP 0 401 384; WO92/16221; WO 95/34326; WO 95/13312; WO 96/11953; PCT InternationalApplication No. US96/19459; and the other publications cited herein thatrelate to pegylation, the disclosures of which are hereby incorporatedby reference.

A specific embodiment of the present invention is an unbranchedmonomethoxy-polyethylene glycol aldehyde molecule having an averagemolecular weight of about 20 kDa conjugated via reductive alkylation tothe N-terminus of a KGF-2 protein(s).

Polyvalent Forms

Polyvalent forms, i.e., molecules comprising more than one activemoiety, may be constructed. In one embodiment, the molecule may possessmultiple KGF-2 protein(s). Additionally, the molecule may possess atleast one KGF-2 protein(s) and, depending upon the desiredcharacteristic of polyvalent form, at least one other molecule.

In one embodiment, KGF-2 protein(s) may be chemically coupled. Forexample, KGF-2 protein(s) may be chemically coupled to a divalent watersoluble molecule via the pegylation technology described above.Additionally, KGF-2 protein(s) may be chemically coupled to biotin, andthe biotin/KGF-2 protein(s) which are conjugated are then allowed tobind to avidin, resulting in tetravalent avidin/biotin/KGF-2 protein(s).KGF-2 protein(s) may also be covalently coupled to dinitrophenol (DNP)or trinitrophenol (TNP) and the resulting conjugates precipitated withanti-DNP or anti-TNP-IgM to form decameric conjugates.

In yet another embodiment, a recombinant fusion protein may also beproduced having KGF-2 protein(s) wherein each recombinant chimericmolecule has a KGF-2 protein(s) sequence, as described above,substituted for the variable domains of either or both of theimmunoglobulin molecule heavy and light chains and having all or partsof the constant domains, but at least one constant domain, of the heavyor light chain of human immunoglobulin. For example, each such chimericKGF-2 protein(s)/IgG1 fusion protein may be produced from two chimericgenes: KGF-2 protein(s)/human kappa light chain chimera (KGF-2protein(s)/Ck) and KGF-2 protein(s)/human gamma-1 heavy chain chimera(KGF-2 protein(s)/Cg-1). Following transcription and translation of thetwo chimeric genes, as described below, the gene products may beassembled into a single chimeric molecule having a KGF-2 protein(s)displayed bivalently. Additional details relating to the construction ofsuch chimeric molecules are disclosed in U.S. Pat. No. 5,116,964, PCTPublication No. WO 89/09622, PCT Publication No. WO 91/16437 and EP315062, the disclosures of which are hereby incorporated by reference.

In yet a further embodiment, recombinant fusion proteins may also beproduced wherein each recombinant chimeric molecule has at least oneKGF-2 protein(s), as described above, and at least a portion of theregion 186-401 of osteoprotegerin (OPG), as described in European PatentApplication No. 96309363.8.

The production of KGF-2 protein(s) are described in further detailbelow. Such proteins may be prepared, for example, by recombinanttechniques or by in vitro chemical synthesis of the desired KGF-2protein(s).

Polynucleotides

Based upon the present description and using the universal codon table,one of ordinary skill in the art can readily determine all of thenucleic acid sequence which encodes an amino acid sequence of a KGF-2protein(s).

Recombinant expression techniques conducted in accordance with thedescriptions set forth below may be followed to produce thesepolynucleotides to express the encoded proteins. For example, byinserting a nucleic acid sequence which encodes a KGF-2 protein(s) intoan appropriate vector, one skilled in the art can readily produce largequantities of the desired nucleotide sequence. The sequences can then beused to generate detection probes or amplification primers.Alternatively, a polynucleotide encoding a KGF-2 protein(s) can beinserted into an expression vector. By introducing the expression vectorinto an appropriate host, the desired KGF-2 protein(s) may be producedin large amounts.

As further described herein, there are numerous host/vector systemsavailable for the propagation of desired nucleic acid sequences and/orthe production of KGF-2 protein(s). These include, but are not limitedto, plasmid, viral,and insertional vectors, and prokaryotic andeukaryotic hosts. One skilled in the art can adapt a host/vector systemwhich is capable of propagating or expressing heterologous DNA toproduce or express the sequences of the present invention.

Furthermore, it will be appreciated by those skilled in the art that, inview of the present disclosure, the nucleic acid sequences include thenucleic acids 109 to 624 of SEQ ID NO:1, as well as degenerate nucleicacid sequences thereof, nucleic acid sequences which encode variant(s)of mature KGF-2 and those nucleic acid sequences which hybridize (underhybridization conditions disclosed in the cDNA library screening sectionbelow, or equivalent conditions or more stringent conditions) tocomplements of nucleic acids 109 to 624 of SEQ ID NO:1.

Also provided by the present invention are recombinant DNA constructsinvolving vector DNA together with the DNA sequences encoding KGF-2protein(s). In each such DNA construct, the nucleic acid sequenceencoding a KGF-2 protein(s) (with or without signal peptides) is inoperative association with a suitable expression control or regulatorysequence capable of directing the replication and/or expression of theKGF-2 protein(s) in a selected host.

Preparation of Polynucleotides

A nucleic acid sequence encoding a KGF-2 protein(s) can readily beobtained in a variety of ways including, without limitation, chemicalsynthesis, cDNA or genomic library screening, expression libraryscreening, and/or PCR amplification of cDNA. These methods and otherswhich are useful for isolating such nucleic acid sequences are set forthin Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;

by Ausubel et al. (1994), eds Current Protocols in Molecular Biology,Current Protocols Press; and by Berger and Kimmel (1987), Methods inEnzymology: Guide to Molecular Cloning Techniques, Vol. 152, AcademicPress, Inc., San Diego, Calif., the disclosures of which are herebyincorporated by reference.

Chemical synthesis of nucleic acid sequences which encode desiredproteins can be accomplished using methods well known in the art, suchas those set forth by Engels et al. (1989), Angew. Chem. Intl. Ed.,28:716-734 and Wells et al. (1985), Gene, 34:315, the disclosures ofwhich are hereby incorporated by reference. These methods include, interalia, the phosphotriester, phosphoramidite and H-phosphonate methods ofnucleic acid sequence synthesis. Large nucleic acid sequences, forexample those larger than about 100 nucleotides in length, can besynthesized as several fragments. The fragments can then be ligatedtogether to form a suitable nucleic acid sequence. A preferred method ispolymer-supported synthesis using standard phosphoramidite chemistry.

Alternatively, a suitable nucleic acid sequence may be obtained byscreening an appropriate cDNA library (i.e., a library prepared from oneor more tissue sources believed to express the protein) or a genomiclibrary (a library prepared from total genomic DNA). The source of thecDNA library is typically a tissue from any species that is believed toexpress a desired protein in reasonable quantities. The source of thegenomic library may be any tissue or tissues from any mammalian or otherspecies believed to harbor a gene encoding a KGF-2 protein(s).

Hybridization mediums can be screened for the presence of a DNA encodinga KGF-2 protein(s) using one or more nucleic acid probes(oligonucleotides, cDNA or genomic DNA fragments that possess anacceptable level of homology to the cDNA or gene to be cloned) that willhybridize selectively with cDNA(s) or gene(s) present in the library.The probes typically used for such screening encode a small region ofDNA sequence from the same or a similar species as the species fromwhich the library is prepared. Alternatively, the probes may bedegenerate, as discussed herein.

Hybridization is typically accomplished by annealing the oligonucleotideprobe or cDNA to the clones under conditions of stringency that preventnon-specific binding but permit binding of those clones that have asignificant level of homology with the probe or primer. Typicalhybridization and washing stringency conditions depend in part on thesize (i.e., number of nucleotides in length) of the cDNA oroligonucleotide probe and whether the probe is degenerate. Theprobability of identifying a clone is also considered in designing thehybridization medium (e.g., whether a cDNA or genomic library is beingscreened).

Where a DNA fragment (such as cDNA) is used as a probe, typicalhybridization conditions include those as set forth in Ausubel et al.(1994), eds., supra. After hybridization, the hybridization medium iswashed at a suitable stringency, depending on several factors such asprobe size, expected homology of probe to clone, the hybridizationmedium being screened, the number of clones being screened, and thelike. Exemplary stringent hybridization conditions are hybridization in4×SSC at 62-67° C., followed by washing in 0.1×SSC at 62-67° C. forapproximately an hour. Alternatively, exemplary stringent hybridizationconditions are hybridization in 45-55% formamide, 4×SSC at 40-45° C.Also included are DNA sequences which hybridize to the nucleic acidsequences set forth in FIG. 1 under relaxed hybridization conditions andwhich encode KGF-2 protein(s). Examples of such relaxed stringencyhybridization conditions are 4×SSC at 45-55° C. or hybridization with30-40% formamide at 40-45° C. See Maniatis et al. (1982), MolecularCloning (A Laboratory Manual), Cold Spring Harbor Laboratory, pages 387to 389.

There are also exemplary protocols for stringent washing conditionswhere oligonucleotide probes are used to screen hybridization mediums.For example, a first protocol uses 6×SSC with 0.05 percent sodiumpyrophosphate at a temperature of between about 35° C. and 63° C.,depending on the length of the probe. For example, 14 base probes arewashed at 35-40° C., 17 base probes at 45-50° C., 20 base probes at52-57° C., and 23 base probes at 57-63° C. The temperature can beincreased 2-3° C. where the background non-specific binding appearshigh. A second protocol uses tetramethylammonium chloride (TMAC) forwashing. One such stringent washing solution is 3 M TMAC, 50 mMTris-HCl, pH 8.0 and 0.2% SDS.

Another method for obtaining a suitable nucleic acid sequence is thepolymerase chain reaction (PCR). In this method, cDNA is prepared frompoly(A)+RNA or total RNA using the enzyme reverse transcriptase. Twoprimers, typically complementary to two separate regions of cDNA(oligonucleotides) encoding a KGF-2 protein(s), are then added to thecDNA along with a polymerase such as Taq polymerase, and the polymeraseamplifies the cDNA region between the two primers.

The oligonucleotide sequences selected as probes or primers should be ofadequate length and sufficiently unambiguous so as to minimize theamount of non-specific binding that may occur during screening or PCRamplification. The actual sequence of the probes or primers is usuallybased on conserved or highly homologous sequences or regions.Optionally, the probes or primers can be fully or partially degenerate,i.e., can contain a mixture of probes/primers, all encoding the sameamino acid sequence but using different codons to do so. An alternativeto preparing degenerate probes is to place an inosine in some or all ofthose codon positions that vary by species. The oligonucleotide probesor primers may be prepared by chemical synthesis methods for DNA, asdescribed above.

Vectors

DNA encoding a KGF-2 protein(s) may be inserted into vectors for furthercloning (amplification of the DNA) or for expression. Suitable vectorsare commercially available, or the vector may be specificallyconstructed. The selection or construction of an appropriate vector willdepend on (1) whether it is to be used for DNA amplification or for DNAexpression, (2) the size of the DNA to be inserted into the vector, and(3) the intended host cell to be transformed with the vector.

The vectors each involve a nucleic acid sequence which encodes a desiredprotein operatively linked to one or more of the following expression.control or regulatory sequences capable of directing, controlling orotherwise affecting the expression of a desired protein by a selectedhost cell. Each vector contains various components, depending on itsfunction (amplification of DNA or expression of DNA) and itscompatibility with the intended host cell. The vector componentsgenerally include, but are not limited to, one or more of the following:a signal sequence, an origin of replication, one or more selection ormarker genes, promoters, enhancer elements, a transcription, terminationsequence and the like. These components may be obtained from naturalsources or be synthesized by known procedures.

Examples of suitable prokaryotic cloning vectors include bacteriophages,such as lambda derivatives, or plasmids from E. coli (e.g. pBR322, colE1, pUC, the F-factor and Bluescript® plasmid derivatives (Stratagene,LaJolla, Calif.)). Other appropriate expression vectors, of whichnumerous types are known in the art for the host cells described below,can also be used for this purpose.

Signal Sequence

The nucleic acid encoding a signal sequence may be inserted 5′ of thesequence encoding a desired protein, e.g, it may be a component of avector or it may be a part of a nucleic acid encoding a desired protein.The nucleic acid encoding the native signal sequence of KGF-2 protein(s)is known (WO 96/25422).

Origin of Replication

Expression and cloning vectors each generally include a nucleic acidsequence that enables the vector to replicate in one or more selectedhost cells. In a cloning vector, this sequence is typically one thatenables the vector to replicate independently of the host chromosomalDNA and includes origins of replication or autonomously replicatingsequences. Such sequences are well known. The origin of replication fromthe plasmid pBR322 is suitable for most Gram-negative bacteria, andvarious origins (e.g., SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells. Generally, the origin ofreplication is not needed for mammalian expression vectors (for example,the SV40 origin is often used only because it contains the earlypromoter).

Selection Gene

The expression and cloning vectors each typically contain a selectiongene. This gene encodes a “marker” protein necessary for the survival orgrowth of the transformed host cells when grown in a selective culturemedium. Host cells that are not transformed with the vector will notcontain the selection gene and, therefore, they will not survive in theculture medium. Typical selection genes encode proteins that (a) conferresistance to antibiotics or other toxins, e.g., ampicillin, neomycin,methotrexate or tetracycline; (b) complement auxotrophic deficiencies;or (c) supply critical nutrients not available from the culture medium.

Other selection genes may be used to amplify the genes to be expressed.Amplification is the process wherein genes which are in greater demandfor the production of a protein critical for growth are reiterated intandem within the chromosomes of successive generations of recombinantcells. Examples of suitable selectable markers for mammalian cellsinclude dihydrofolate reductase (DHFR) and thymidine kinase. The celltransformants are placed under selection pressure which only thetransformants are uniquely adapted to survive by virtue of the markerpresent in the vector. Selection pressure is imposed by culturing thetransformed cells under conditions in which the concentration ofselection agent in the medium is successively changed, thereby leadingto amplification of both the selection gene and the DNA that encodes thedesired protein. As a result, increased quantities of the desiredprotein are synthesized from the amplified DNA.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate, a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is used is the Chinese hamsterovary cell line deficient in DHFR activity (Urlaub and Chasin (1980),Proc. Natl. Acad. Sci., USA, 77(7):4216-4220, the disclosure of which ishereby incorporated by reference). The transformed cells are thenexposed to increased levels of methotrexate. This leads to the synthesisof multiple copies of the DHFR gene and, concomitantly, multiple copiesof other DNA present in the expression vector, such as the DNA encodingthe desired protein.

Promoter

Expression and cloning vectors each will typically contain a promoterthat is recognized by the host organism and is operably linked to anucleic acid sequence encoding the desired protein. A promoter is anuntranslated sequence located upstream (5′) to the start codon of astructural gene (generally within about 100 to 1000 bp) that controlsthe transcription and translation of a particular nucleic acid sequence.A promoter may be conventionally grouped into one of two classes,inducible promoters and constitutive promoters. An inducible promoterinitiates increased levels of transcription from DNA under its controlin response to some change in culture conditions, such as the presenceor absence of a nutrient or a change in temperature. A large number ofpromoters, recognized by a variety of potential host cells, are wellknown. A promoter may be operably linked to DNA encoding the desiredprotein by removing the promoter from the source DNA by restrictionenzyme digestion and inserting the desired promoter sequence. The nativeKGF-2 promoter sequence may be used to direct amplification and/orexpression of DNA encoding a desired protein. A heterologous promoter ispreferred, however, if it permits greater transcription and higheryields of the expressed protein as compared to the native promoter andif it is compatible with the host cell system that has been selected foruse. For example, any one of the native promoter sequences of other FGFfamily members may be used to direct amplification and/or expression ofthe DNA encoding a desired protein.

Promoters suitable for use with prokaryotic hosts include thebeta-lactamase and lactose promoter systems; alkaline phosphatase, atryptophan (trp) promoter system; a bacterial luminescence (luxR) genesystem; and hybrid promoters such as the tac promoter. Other knownbacterial promoters are also suitable. Their nucleotide sequences havebeen published, thereby enabling one skilled in the art to ligate themto the desired DNA sequence(s) using linkers or adaptors as needed tosupply any required restriction sites.

Suitable promoting sequences for use with yeast hosts are also wellknown in the art. Suitable promoters for use with mammalian host cellsare well known and include those obtained from the genomes of virusessuch as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2),bovine papilloma virus, avian sarcoma virus, cytomegalovirus, aretrovirus, hepatitis-B virus and, most preferably, Simian Virus 40(SV40). Other suitable mammalian promoters include heterologousmammalian promoters, e.g., heat-shock promoters and the actin promoter.

Enhancer Element

The expression and cloning vectors each will typically contain anenhancer sequence to increase the transcription by higher eukaryotes ofa DNA sequence encoding a desired protein. Enhancers are cis-actingelements of DNA, usually from about 10-300 bp in length, that act on thepromoter to increase its transcription.

Enhancers are relatively orientation and position independent. They havebeen found 5′ and 3′ to the transcription unit. Yeast enhancers areadvantageously used with yeast promoters. Several enhancer sequencesavailable from mammalian genes are known (e.g., globin, elastase,albumin, alpha-feto-protein and insulin). Additionally, viral enhancerssuch as the SV40 enhancer, the cytomegalovirus early promoter enhancer,the polyoma enhancer and adenovirus enhancers are exemplary enhancingelements for the activation of eukaryotic promoters. While an enhancermay be spliced into a vector at a position 5′ or 3′ to a DNA encoding adesired protein, it is typically located at a site 5′ from the promoter.

Transcription Termination

Expression vectors used in eukaryotic host cells each will typicallycontain a sequence necessary for the termination of transcription andfor stabilizing the mRNA. Such sequences are commonly available from the5′ and occasionally 3′ untranslated regions of eukaryotic DNAs or cDNAs.These regions contain nucleotide segments transcribed as polyadenylatedfragments in the untranslated portion of the mRNA encoding a desiredprotein.

The construction of a suitable vector containing one or more of theabove-listed components (together with the desired coding sequence) isaccomplished by standard ligation techniques. Isolated plasmids or DNAfragments are cleaved, tailored and religated in the desired order togenerate the required vector. To confirm that the correct sequence hasbeen constructed, the ligation mixture may be used to transform E. coli,and successful transformants may be selected by known techniques asdescribed above. Quantities of the vector from the transformants arethen prepared, analyzed by restriction endonuclease digestion, and/orsequenced to confirm the presence of the desired construct.

A vector that provides for the transient expression of DNA encoding adesired protein in mammalian cells may also be used. In general,transient expression involves the use of an expression vector that isable to replicate efficiently in a host cell, such that the host cellaccumulates many copies of the expression vector and, in turn,synthesizes high levels of the desired protein encoded by the expressionvector. Each transient expression systems, comprising a suitableexpression vector and a host cell, allows for the convenient positiveidentification of proteins encoded by cloned DNAs, as well as for therapid screening of such proteins for desired biological or physiologicalproperties.

Host Cells

Any of a variety of recombinant host cells, each of which contains anucleic acid sequence for use in expressing a desired protein, is alsoprovided by the present invention. Exemplary prokaryotic and eukaryotichost cells include bacterial, mammalian, fungal, insect, yeast or plantcells.

Prokaryotic host cells include but are not limited to eubacteria such asGram-negative or Gram-positive organisms (e.g., E. coli (HB101, DH5a,DH10 and MC1061); Bacilli such as B. subtilis; Pseudomonas species, suchas P. aeruginosa; Streptomyces spp.; Salmonella typhimurium; or Serratiamarcescans. As a specific embodiment, a KGF-2 protein(s) may beexpressed in E. coli.

In addition to prokaryotic host cells, a KGF protein(s) may be expressedin glycosylated form by any one of a number of suitable host cellsderived from multicellular organisms. Such host cells are capable ofcomplex processing and glycosylation activities. In principle, anyhigher eukaryotic cell culture might be used, whether such cultureinvolves vertebrate or invertebrate cells, including plant and insectcells.

Eukaryotic microbes such as filamentous fungi or yeast may be suitablehosts for the expression of a KGF-2 protein(s). Saccharomycescerevisiae, or common baker's yeast, is the most commonly used amonglower eukaryotic host microorganisms, but a number of other genera,species and strains are well known and commonly available.

Vertebrate cells may be used, as the propagation of vertebrate cells inculture (tissue culture) is a well-known procedure. Examples of usefulmammalian host cell lines include but are not limited to monkey kidneyCV1 line transformed by SV40 (COS-7), human embryonic kidney line (293cells or 293 cells subcloned for growth in suspension culture), babyhamster kidney cells and Chinese hamster ovary cells. Other suitablemammalian cell lines include but are not limited to HeLa, mouse L-929cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, and BHK or HaKhamster cell lines. As a specific embodiment, a KGF-2 protein(s) may beexpressed in COS cells or in baculovirus cells.

A host cell may be transfected and preferably transformed with a desirednucleic acid under appropriate conditions permitting expression of thenucleic acid. The selection of suitable host cells and methods fortransformation, culture, amplification, screening and product productionand purification are well known in the art (Gething and Sambrook (1981),Nature, 293:620-625 or, alternatively, Kaufman et al. (1985), Mol. Cell.Biol., 5(7):1750-1759, or U.S. Pat. No. 4,419,446, the disclosures ofwhich are hereby incorporated by reference). For example, for mammaliancells without cell walls, the calcium phosphate precipitation method maybe used. Electroporation, microinjection and other known techniques mayalso be used.

It is also possible that a desired protein may be produced by homologousrecombination or with recombinant production methods utilizing controlelements introduced into cells already containing MA encoding a KGF-2protein(s). Homologous recombination is a technique originally developedfor targeting genes to induce or correct mutations in transcriptionallyactive genes (Kucherlapati (1989), Prog. in Nucl. Acid Res. and Mol.Biol., 36:301, the disclosure of which is hereby incorporated byreference). The basic technique was developed as a method forintroducing specific mutations into specific regions of the mammaliangenome (Thomas et al. (1986), Cell, 44:419-428; Thomas and Capecchi(1987), Cell, 51:503-512 and Doetschman et al. (1988), Proc. Natl. Acad.Sci., 85:8583-8587, the disclosures of which are hereby incorporated byreference) or to correct specific mutations within defective genes(Doetschman et al. (1987), Nature, 330:576-578, the disclosure of whichis hereby incorporated by reference). Exemplary techniques are describedin U.S. Pat. No. 5,272,071; WO 92/01069; WO 93/03183; WO 94/12650 and WO94/31560, the disclosures of which are hereby incorporated by reference.

Culturing the Host Cells

The method for culturing each of the one or more recombinant host cellsfor production of a desired protein will vary depending upon manyfactors and considerations; the optimum production procedure for a givensituation will be apparent to those skilled in the art through minimalexperimentation. Such recombinant host cells are cultured in suitablemedium and the expressed protein is then optionally recovered, isolatedand purified from the culture medium (or from the cell, if expressedintracellularly) by appropriate means known to those skilled in the art.

Specifically, each of the recombinant cells used to produce a desiredprotein may be cultured in media suitable for inducing promoters,selecting suitable recombinant host cells or amplifying the geneencoding the desired protein. The media may be supplemented as necessarywith hormones and/or other growth factors (such as insulin, transferrinor epidermal growth factor), salts (such as sodium chloride, calcium,magnesium and phosphate), buffers (such as HEPES), nucleosides (such asadenosine and thymidine), antibiotics (such as gentamicin), traceelements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range), and glucose or another energysource. Other supplements may also be included, at appropriateconcentrations, as will be appreciated by those skilled in the art.Suitable culture conditions, such as temperature, pH and the like, arealso well known to those skilled in the art for use with the selectedhost cells.

The resulting expression product may then be purified to nearhomogeneity using procedures known in the art. Exemplary purificationtechniques are taught in published PCT Application Nos. WO 90/08771 andWO 96/11952, the disclosures of which are hereby incorporated byreference.

Uses

Variant(s) of KGF-2 described herein, and chemically-modifiedderivatives of KGF-2 and variant(s) of KGF-2 protein (collectively,“KGF-2 protein product(s)”) may be used as research reagents and astherapeutic and diagnostic agents. Thus, a KGF-2 protein product(s) maybe used in in vitro and/or in vivo diagnostic assays to quantify theamount of KGF-2 in a tissue or organ sample.

For example, a KGF-2 protein product(s) can be used for identificationof the receptor(s) for a KGF-2 protein(s) in various body fluids andtissue samples using techniques known in the art (WO 90/08771).

This invention also contemplates the use of a KGF-2 protein product(s)in the generation of antibodies made against the KGF-2 proteinproduct(s), including native KGF-2. One of ordinary skill in the art canuse well-known, published procedures to obtain monoclonal and polyclonalantibodies or recombinant antibodies. Such antibodies may then be usedto purify and characterize KGF-2 protein product(s), including nativeKGF-2.

Pharmaceutical Compositions

The present invention encompasses pharmaceutical preparations eachcontaining therapeutically- or prophylatically-effective amounts of aKGF-2 protein product(s).

Pharmaceutical compositions each will generally include atherapeutically-effective or prophylatically-effective amount of a KGF-2protein product(s) in admixture with a vehicle. The vehicle preferablyincludes one or more pharmaceutically and physiologically acceptableformulation materials in admixture with the KGF-2 protein product(s).

The primary solvent in a vehicle may be either aqueous or non-aqueous innature. In addition, the vehicle may contain other pharmaceuticallyacceptable excipients for modifying or maintaining the pH (e.g., bufferssuch as citrates, phosphates, and amino acids such as glycine);osmolarity (e.g., mannitol and sodium chloride); viscosity; clarity;color; sterility; stability (e.g., sucrose and sorbitol); odor of theformulation; rate of dissolution (e.g., solubilizers or solubilizingagents such as alcohols, polyethylene glycols and sodium chloride); rateof release; as well as bulking agents for lyophilized formulation (e.g.,mannitol and glycine); surfactants (e.g., polysorbate 20, polysorbate80, triton, and pluronics); antioxidants (e.g., sodium sulfite andsodium hydrogen-sulfite); preservatives (e.g., benzoic acid andsalicylic acid); flavoring and diluting agents; emulsifying agents;suspending agents; solvents; fillers; delivery vehicles; diluents and/orpharmaceutical adjuvants. Other effective administration forms such asparenteral slow-release formulations, inhalant mists, orally-activeformulations, or suppositories are also envisioned.

The composition may also involve particulate preparations of polymericcompounds such as bulk erosion polymers (e.g., poly(lactic-co-glycolicacid) (PLGA) copolymers, PLGA polymer blends, block copolymers of PEG,and lactic and glycolic acid, poly(cyanoacrylates)); surface erosionpolymers (e.g., poly(anhydrides) and poly(ortho esters)); hydrogelesters (e.g., pluronic polyols, poly(vinyl alcohol),poly(vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers,cellulose, hyaluronic acid derivatives, alginate, collagen, gelatin,albumin, and starches and dextrans) and composition systems thereof; orpreparations of liposomes or microspheres. Such compositions mayinfluence the physical state, stability, rate of in vivo release, andrate of in vivo clearance of the present proteins and derivatives. Theoptimal pharmaceutical formulation for a desired protein will bedetermined by one skilled in the art depending upon the route ofadministration and desired dosage. Exemplary pharmaceutical compositionsare disclosed in Remington's Pharmaceutical Sciences, 18th Ed. (1990),Mack Publishing Co., Easton, Pa. 18042, pages 1435-1712; Gombotz andPettit (1995), Bioconjugate Chem., 6:332-351; Leone-Bay, et al. (1995),Journal of Medicinal Chemistry, 38:4263-4269; Haas, et al. (1995),Clinical Immunology and Immunopathology, 76(1):93; WO 94/06457; WO94/21275; FR 2706772 and WO 94/21235, the disclosures of which areincorporated herein by reference.

Specific sustained release compositions are available from the a varietyof suppliers including Depotech Corp. (Depofoam™, a multivesicularliposome); Alkermes, Inc. (ProLease™, a PLGA microsphere). As usedherein, hyaluronan is intended to include hyaluronan, hyaluronic acid,salts thereof (such as sodium hyaluronate), esters, ethers, enzymaticderivatives and cross-linked gels of hyaluronic acid, and chemicallymodified derivatives of hyaluronic acid (such as hylan). Exemplary formsof hyaluronan are disclosed in U.S. Pat. Nos. 4,582,865, 4,605,691,4,636,524, 4,713,448, 4,716,154, 4,716,224, 4,772,419, 4,851,521,4,957,774, 4,863,907, 5,128,326, 5,202,431, 5,336,767, 5,356,883;European Patent Application Nos. 0 507 604 A2 and 0 718 312 A2; and WO96/05845, the disclosures of which are hereby incorporated by reference.Suppliers of hyaluronan include Biomatrix, Inc. Ridgefield, N.J.; FidiaS.p.A., Abano Terme, Italy; Kaken Pharmaceutical Co., Ltd., Tokyo,Japan; Pharmacia AB, Stockholm, Sweden; Genzyme Corporation, Cambridge,Mass.; Pronova Biopolymer, Inc. Portsmouth, N.H.; Calbiochem-NovabiochemAB, Lautelfingen, Switzerland; Intergen Company, Purchase, N.Y. andKyowa Hakko Kogyo Co., Ltd., Tokyo, Japan.

For treatment and/or prevention of oral indications, a liquid solutionor suspension can be used in a manner similar to a mouthwash, where theliquid is swished around in the mouth so as to maximize treatment oflesions (U.S. Pat. No. 5,102,870, the teachings of which areincorporated by reference). Longer contact with the mucosal surface canbe attained by selecting a suitable vehicle which is capable of coatingmucosa. Typical examples are pectin containing formulations such asOrabase Registered™ (Colgate-Hoyt Laboratories, Norwood, Mass.),sucralfate suspensions, Kaopectate and Milk of Magnesia. The formulationcan also be a spreadable cream, gel, lotion or ointment having apharmaceutically acceptable non-toxic vehicle or carrier. KGF-2 proteinproduct(s) can also be incorporated into a slow dissolving lozenge ortroche, a chewing gum base, or a buccal or slow delivery prosthesishooked onto a back molar, for example. Therapeutic agents such asanalgesics and anesthetics can be administered to alleviate pain andsuch as anti-infectictives, anti-bacterials, anti-fungals andantiseptics can be administered to prevent and/or treat secondaryinfection of the lesions.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or a dehydrated or lyophilized powder. Such formulations may be storedeither in a ready-to-use form or in a form (e.g., lyophilized) requiringreconstitution prior to administration.

In a specific embodiment, the present invention is directed to kits forproducing a single-dose administration unit. The kits may each containboth a first container having a dried protein and a second containerhaving an aqueous formulation. Kits included within the scope of thisinvention are single and multi-chambered pre-filled syringes; exemplarypre-filled syringes (e.g., liquid syringes, and lyosyringes such asLyo-Ject®, a dual-chamber pre-filled lyosyringe) are available fromVetter GmbH, Ravensburg, Germany.

It should be noted that KGF-2 protein product(s) formulations describedherein may be used for veterinary as well as human applications and thatthe term “patient” should not be construed in a limiting manner.

The frequency of dosing the KGF-2 protein product(s) to a patient willdepend on the disease and the condition of the patient, as well as thepharmacokinetic parameters of KGF-2 protein product(s) as formulated,and the route of administration. The KGF-2 protein product(s) may beadministered once, administered daily, or administered with an initialbolus dose followed by a continuous dose or sustained delivery. It isalso contemplated that other modes of a continuous or near-continuousdosing may be practiced. For example, chemical derivatization may resultin sustained release forms of the protein which have the effect of acontinuous presence in the bloodstream, in predictable amounts, based ona determined dosage regimen.

A patient in need of stimulation (including cytoprotection,proliferation and/or differentiation) of epithelial cells may beadministered an effective amount of a KGF-2 protein product(s) to elicitthe desired response in the patient and will, thus, generally bedetermined by the attending physician. The dosage regimen involved in amethod of preventing or treating a specific condition will be determinedby the attending physician, considering various factors which modify theaction of drugs, e.g., the age, condition, body weight, sex and diet ofthe patient, the severity of any infection, the time of administrationand other clinical factors. Appropriate dosages may be ascertainedthrough use of established assays for determining dosages utilized inconjunction with appropriate dose-response data. Typical dosages willrange from 0.001 mg/kg body weight to 500 mg/kg body weight, preferablyup to 200 mg/kg body weight, more preferably 100 mg/kg body weight.

The KGF-2 protein product(s) may be administered via topical, enteral orparenteral administration including, without limitation, intravenous,intramuscular, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,and intrasternal injection and infusion. The KGF-2 protein product(s)may be administered via oral administration or administered throughmucus membranes, that is, intranasally, sublingually, buccally orrectally for systemic delivery. The KGF-2 protein product(s) may be usedonce or administered repeatedly, depending on the disease and thecondition of the patient. In some cases, the KGF-2 protein product(s)may be administered as an adjunct to other therapy and also with otherpharmaceutical preparations

In another embodiment, cell therapy (e.g., implantation of cellsproducing KGF-2 protein(s) is also contemplated. This embodiment of thepresent invention may include implanting into patients cells which arecapable of synthesizing and secreting a biologically-active form ofKGF-2 protein(s). Such cells producing KGF-2 protein(s) may be cellswhich do not normally produce KGF-2 protein(s) but which have beenmodified to produce KGF-2 protein(s), or which may be cells whoseability to produce KGF-2 protein(s) have been augmented bytransformation with a polynucleotide suitable for the expression andsecretion of such protein. In order to minimize a potentialimmunological reaction in patients being administered KGF-2 protein(s)of a foreign species, it is preferred that the cells be of the samespecies as the patient (e.g., human) or that the cells may beencapsulated with material that provides a barrier against immunerecognition, or that cells be placed into an immunologically-privilegedanatomical location, such as in the testis, eye and central nervoussystem.

Human or non-human animal cells may be implanted in patients inbiocompatible, semi-permeable polymeric enclosures or membranes to allowrelease of a KGF-2 protein(s), but prevent destruction of the cells bythe patient's immune system or by other detrimental factors from thesurrounding tissue. Alternatively, the patient's own cells, transformedex vivo to produce KGF-2 protein(s), could be implanted directly intothe patient without such encapsulation. The methodology for the membraneencapsulation of living cells is familiar to those of ordinary skill inthe art, and the preparation of the encapsulated cells and theirimplantation in patients may be accomplished with known techniques (U.S.Pat. Nos. 4,892,538; 5,011,472; and 5,106,627, the disclosures of whichare hereby incorporated by reference).

In yet another embodiment, in vivo gene therapy is also envisioned,wherein a nucleic acid sequence encoding a KGF-2 protein(s) isintroduced directly into a patient. Efficient and long lasting genetransfer to hepatocytes is required for effective gene therapy for localexpression of the protein to prevent and/or treat liver diseases and/orfor secretion of the protein to prevent and/or treat diseases in otherorgans or tissues.

The DNA construct may be directly injected into the tissue of the organto be treated, where it can be taken up in vivo and expressed, providedthat the DNA is operable linked to a promoter that is active in suchtissue. The DNA construct may also additionally include vector sequencefrom such vectors as an adenovirus vector, a retroviral vector,papilloma virus and/or a herpes virus vector, to aid uptake in thecells. Physical transfer may be achieved in vivo by local injection ofthe desired nucleic acid construct or other appropriate delivery vectorcontaining the desired nucleic acid sequence, such as liposome-mediatedtransfer, direct injection (naked DNA), receptor-mediated transfer(ligand-DNA complex), or microparticle bombardment (gene gun). For thein vivo regeneration of hepatocytes in the liver, the use of Moloneyretroviral vectors may be especially effective (Bosch, et al. (1996),Cold Spring Harbor, Gene Therapy Meeting, Sep. 25-29, 1996; and Bosch,et al. (1996), Journal of Clinical Investigation, 98(12):2683-2687).

A KGF-2 protein product(s) may be applied in therapeutically- andprophylactically-effective amounts to organs or tissues specificallycharacterized by having damage to or clinically insufficient numbers ofepithelium cells. It should be noted that a KGF-2 protein product(s) maybe used for veterinary as well as human applications and that the term“patient” should not be construed in a limiting manner.

In accordance with the present invention, a KGF-2 protein product(s) maybe used in vivo to induce stimulation (including cytoprotection,proliferation and/or differentiation), proliferation and/ordifferentiation of epithelial cells including, but not limited to, theeye, ear, gums, hair, lung, skin, pancreas (endocrine and exocrine),thymus, thyroid, urinary bladder, liver and gastrointestinal tractincluding cells in the oral cavity, in the esophagus, in the glandularstomach and small intestine, in the colon and the intestinal mucosa, inthe rectum and in the anal canal. Indications in which a KGF-2 proteinproduct(s) may be successfully administered include, but are not limitedto: burns and other partial and full-thickness injuries in need ofstimulation of adnexal structures such as hair follicles, sweat glands,and sebaceous glands; lesions caused by epidermolysis bullosa, which isa defect in adherence of the epidermis to the underlying dermis,resulting in frequent open, painful blisters which can cause severemorbidity; chemotherapy-induced alopecia and male-pattern baldness, orthe progressive loss of hair in men and women; gastric and duodenalulcers; gut toxicity in radiation- and chemotherapy-treatment regimes;erosions of the gastrointestinal tract (e.g., esophagus, stomach andintestines) include erosive gastritis, esophagitis, esophageal reflux orinflammatory bowel diseases, such as Crohn's disease (affectingprimarily the small intestine) and ulcerative colitis (affectingprimarily the large bowel); disorders or damage to salivary gland tissueincluding radiation/chemotherapy effects, autoimmune diseases such asSjogren's Syndrome which can cause salivary gland insufficiency (siccasyndrome); insufficient production of mucus throughout thegastrointestinal tract; adult respiratory distress syndrome (ARDS),pneumonia, hyaline membrane disease (i.e., infant respiratory distresssyndrome and bronchopulmonary dysplasia) in premature infants; acute orchronic lung damage or insufficiency due to inhalation injuries(including high oxygen levels), emphysema, lung damage fromchemotherapeutics, ventilator trauma or other lung damagingcircumstances; hepatic cirrhosis, fulminant liver failure, damage causedby acute viral hepatitis and/or toxic insults to the liver and/or bileduct disorders, and viral-mediated gene transfer to liver; cornealabrasion and/or corneal ulcerations due to chemicals, bacteria orviruses; progressive gum disease; eardrum damage; ulcerations and/orinflammations including conditions resulting from chemotherapy and/orinfection; pancreatic disorders and pancreatic insufficiencies includingdiabetes (Type I and Type II), pancreatitis, cystic fibrosis, and as anadjunct in islet cell transplantation.

This invention thus has significant implications in terms of enablingthe application of KGF-2 protein product(s) specifically characterizedby the prophylactic and/or therapeutic use of KGF-2 to reduce, delayand/or block the onset of damage to or deficiencies in these particulartypes of cells. The following is a more specific description of diseasesand medical conditions which can be treated with KGF-2 proteinproduct(s) in accordance with the invention.

Specific uses of the KGF-2 protein products) are disclosed in patentapplication Ser. No. 09/284,101, filed on the same date herewith byLacey, Ulich, Danilenko and Farrell, entitled on the Applicationtransmittal letter as “USES OF KERATINOCYTE GROWTH FACTOR-2”, thedisclosure of which is hereby incorporated by reference.

KGF-2 protein product(s) are useful to increase cytoprotection,proliferation and/or differentiation of hepatocytes in order to increaseliver-function. KGF-2 protein product(s) are useful to treat and/orprevent hepatic cirrhosis, fulminant liver failure, damage caused byacute viral hepatitis, toxic insults to the liver and/or bile ductdisorders.

Hepatic cirrhosis, secondary to viral hepatitis and chronic alcoholingestion, is a significant cause of morbidity and mortality. KGF-2protein product(s) are useful to treat and/or prevent the development ofcirrhosis. A standard in vivo model of hepatic cirrhosis is known(Tomaszewski et al. (1991), J. Appl. Toxicol., 11:229-231, thedisclosure of which is hereby incorporated by reference).

Fulminant liver failure is a life-threatening condition which occurswith end-stage cirrhosis and which is presently treatable only withliver transplantation. KGF-2 protein product(s) are useful to treatand/or prevent fulminant liver failure. Standard in vivo models offulminant liver failure are known (Mitchell et al. (1973), J. Pharmacol.Exp. Ther., 187:185-194; Thakore and Mehendale (1991), ToxicologicPathol., 19:47-58; and Havill et al. (1994), FASEB Journal, 8(4-5):A930,Abstract 5387, the disclosures of which are hereby incorporated byreference).

Acute viral hepatitis is frequently subclinical and self-limiting.However, in a minority of patients severe liver damage can result overseveral weeks. KGF-2 protein product(s) are useful in preventing and/ortreating viral hepatitis. Standard in vivo models of hepatocyteproliferation are known (Housley et al. (1994), Journal of ClinicalInvestigation, 94(5):1764-1777; and Havill et al. (1994), supra, thedisclosures of which are hereby incorporated by reference).

Toxic insults to the liver caused by acetaminophen, halothane, carbontetrachloride and other toxins may be prevented and/or treated by KGF-2protein product(s). Standard in vivo models of liver toxicity are known(Mitchell et al. (1973), supra; Thakore and Mehendale (1991), supra; andHavill et al. (1994), supra, the disclosures of which are herebyincorporated by reference).

KGF-2 protein product(s) are useful to increase cytoprotection,proliferation and/or differentiation of epithelial cells in thegastrointestinal tract (e.g., the oral cavity, esophagus, stomach, smallintestine, colon, rectum and anal canal). The terms “gastrointestinaltract”, as defined herein, and “gut” are art-recognized terms and areused interchangeably herein. Specifically, KGF-2 protein product(s) areuseful to treat and/or prevent gastric ulcers, duodenal ulcers,inflammatory bowel disease, gut toxicity and erosions of thegastrointestinal tract.

Gastric ulcers cause significant morbidity, have a relatively highrecurrence rate, and heal by scar formation on the mucosal lining. KGF-2protein product(s) are useful to prevent degeneration of glandularmucosa and to regenerate glandular mucosa more rapidly, e.g., offering asignificant therapeutic improvement in the treatment of gastric ulcers.Standard in vivo models of gastric ulcers are known (Tarnawski et al.(1991), “Indomethacin Impairs Quality of Experimental Gastric UlcerHealing: A Quantitative Histological and Ultrastructural Analysis”,In:Mechanisms of Injury, Protection and Repair of the UpperGastrointestinal Tract, (eds) Garner and O'Brien, Wiley & Sons; Brodie(1968), Gastroenterology, 55:25; and Ohning et al. (1994),Gastroenterology, 106(4 Suppl.):A624, the disclosures of which arehereby incorporated by reference).

Duodenal ulcers, like gastric ulcers, cause significant morbidity andhave a relatively high recurrence rate. KGF-2 protein product(s) areuseful to prevent degeneration of the mucosal lining of the duodenum andto rapidly regenerate the mucosal lining of the duodenum to heal thoseulcers and decrease their recurrence. Standard in vivo models ofduodenal ulcers are known (Berg et al. (1949), Proc. Soc. Exp. Biol.Med., 7:374-376; Szabo and Pihan, Chronobiol. Int. (1987), 6:31-42; andRobert et al. (1970), Gastroenterology, 59:95-102, the disclosures ofwhich are hereby incorporated by reference).

Gut toxicity is a major limiting factor associated with cancertreatment, both in radiation (abdominal, total body or local, e.g., headand neck) and chemotherapy. Of primary concern are those patientsundergoing: chemotherapy for cancer such as leukemia, breast cancer oras an adjuvant to tumor removal; radiotherapy for head and neck cancer;and combined chemotherapy and radiotherapy for bone marrow transplants.The severity of damage is related to the type and dose ofchemotherapeutic agent(s) and concomitant therapy such as radiotherapy.

Mucositis in portions of the gastrointestinal tract may account forsignificant pain and discomfort for these patients, and range inseverity from redness and swelling to frank ulcerative lesions. Thelesions often become secondarily infected and become much harder toheal. Standard in vivo models of radiation-induced gut toxicity areknown (Withers and Elkind (1970), Int. J. Radiat., 17(3):261-267, thedisclosure of which is hereby incorporated by reference). Standard invivo models of chemotherapy-induced gut toxicity are known (Farrell etal., The American Society of Hematology, 38th Annual Meeting (Orlando,Fla.), Dec. 6-8, 1996; Sonis et al. (1990), Oral Surg. Oral Med & OralPathol., 69(4):437-443; and Moore (1985), Cancer ChemotherapyPharmacol., 15:11-15, the disclosures of which are hereby incorporatedby reference).

Exemplary chemotherapeutic agents include, but are not limited to, BCNU,busulfan, carboplatin, cyclophosphamide, cisplatin, cytosinearabinoside, daunorubicin, doxorubicin, etoposide, 5-fluorouracil,gemcytabine, ifosphamide, irinotecan, melphalan, methotrexate,navelbine, topotecan, taxol and taxotere, and exemplary treatmentregimes include, but are not limited to, BEAM (busulfan, etoposide,cytosine arabinoside, methotrexate); cyclophosphamide and total bodyirradiation; cyclophosphamide, total body irradiation and etoposide;cyclophosphamide and busulfan; and 5-fluorouracil with leucovorin orlevamisole.

Treatment, pretreatment and/or post-treatment, with KGF-2 proteinproduct(s) are useful to generate a cytoprotective effect orregeneration or both, for example, on the small intestinal mucosa,allowing increased dosages of such therapies while reducing potentialfatal side effects of gut toxicity.

KGF-2 protein product(s) may preferentially be administered in thefollowing settings. Colorectal patients routinely are administered5-fluorouracil with leucovorin on days 1 to 5; KGF-2 protein product(s)may be administered on days −2, −1 and 0. Head and neck cancer patientsroutinely are administered hypofractionated radiation therapy, plus5-fluorouracil and cisplatin over a seven week period; KGF-2 proteinproduct(s) may be administered on days −2, −1 and 0 and thereafter onceper week until the end of the radiation therapy. In lymphomatransplantation patients are frequently administered BEAM therapy for 6days (days 1 to 6); KGF protein product(s) may be administered on days−2, −1 and 0 and as a three day post-treatment (days 7 to 9).

In specific embodiments, KGF-2 protein product(s) may be administeredprophylactically and/or therapeutically to reduce, delay and/or blockthe onset of mucositis (due to chemotherapy and/or radiotherapy), incombination with one or more cytokines to delay and/or block the onsetof cytopenia.

Typically, bone marrow, peripheral blood progenitor cells or stem cells(McNiece et al. (1989), Blood, 74:609-612 and Moore et al. (1979), BloodCells, 5:297-311, the disclosures of which are hereby incorporated byreference) are removed from a patient prior to myelosuppressivecytoreductive therapy (chemotherapy alone or with radiation therapy) andare then readministered to the patient concurrent with or followingcytoreductive therapy in order to counteract the myelosuppressiveeffects of such therapy.

Many different approaches have been undertaken to protect an organismfrom the side effects of radiation or toxic chemicals. One approach isto replace bone marrow cells before toxicity has developed. Anotherapproach is to use progenitor cells from the peripheral blood (PBPC).These PBPC can be collected by apheresis or phlebotomy followingcytokine therapy alone (G-CSF or GM-CSF), or with chemotherapy orcytokines. They can be given back fresh or cryopreserved. If desired,the cells may be CD34+ selected, Tn-cell depleted, tumor cell depleted,or the progenitor cells can be expanded (caused to multiply) by meansknown in the art, prior to administration. The benefits of re-infusionof autologous or allogeneic progenitors following myelosuppressivetherapy have been described in the literature (Morse et al. (1992), Ann.Clin. Lab. Sci., 22:221-225; Kessinger and Armitage (1991), Blood,77:211-213; Kessinger et al. (1989), Blood, 74:1260-1265; Takam et al.(1989), Blood, 74:1245-1251 and Kessinger et al. (1988), Blood,171:723-727).

As used herein, the term “cytokine” is a generic term for proteinsreleased by one cell population which act on another cell asintercellular mediators. Examples of such cytokines are lymphokines,monokines and traditional polypeptide hormones. Included among thecytokines are insulin-like growth factors; human growth hormone;N-methionyl human growth hormone; bovine growth hormone; parathyroidhormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;glycoprotein hormones such as follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH) and leutinizing hormone (LH);hemopoietic growth factor; hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor alpha and-beta; mouse gonadotropin-associated peptide; inhibin; activin; vascularendothelial growth factor; integrin; thrombopoietin; nerve growthfactors such as NGF-beta; platelet-growth factors such as TPO and MGDF;transforming growth factors (TGFs) such as TGF-alpha and TGF-beta;insulin-like growth factor-I and -II; erythropoietin; osteoinductivefactors; interferons such as interferon-alpha, -beta and -gamma; colonystimulating factors (CSFs) such as macrophige-CSF (M-CSF),granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15 and IL-16; andother polypeptide factors. The cytokines can be used alone or incombination to protect against, mitigate and/or reverse myeloid orhematopoietic toxicity associated with cytotoxic agents.

The mode of administration of KGF-2 protein product(s), as well as ofthe cytokine, should be coordinated and optimized. Depending upon thecircumstances, an appropriate dose of KGF-2 protein product(s) can beadministered prior to or subsequent to administration of the therapeuticagent(s). For example, a parameter to be considered is whether thecytokine is administered in a single dose or in multiple doses duringthe course of therapy. Certain cytokines are cleared rapidly from thebody and will require periodic or continuous administration in order fortheir efficacy to be maximized. The manner of administration can differ,depending on whether a pre-treatment or post-treatment of the cytokineis given. For example, if the cytokine is given prior to the cytotoxicagent, it is preferable to administer the cytokine by intravenous bolusinjection for several hours and, optionally, to repeat suchadministration on one or more days during and after completion of thecytotoxic therapy.

In a specific embodiment, KGF-2 protein product(s) are administered(e.g., intraveneously) at 0.1 to 500 micrograms/kg/dose, preferably upto about 200 micrograms/kg/dose, prior to (e.g., 1 to 3 days) and/orafter chemotherapy or radiation therapy, and G-CSF (Neupogen™ orLenograstim™) or GM-CSF (Sargramostim™) is administered (e.g.,subcutaneously) at 5 micrograms/kg/dose for 1 to 10 days (preferably 7to 10 days) after chemotherapy.

Erosions of the gastrointestinal tract (e.g., esophagus, stomach-andintestine) include erosive gastritis, esophagitis, esophageal reflux andinflammatory bowel diseases. Inflammatory bowel diseases, such asCrohn's disease (affecting primarily the small intestine) and ulcerativecolitis (affecting primarily the large bowel), are chronic diseases ofunknown etiology which result in the destruction of the mucosal surface,inflammation, scar and adhesion formation during repair, and significantmorbidity to the affected individuals. KGF-2 protein product(s) areuseful to regenerate the mucosal lining and decrease the recurrence ofthese erosions, resulting in faster healing, and may be of benefit incontrolling progression of the disease. Standard in vivo models oferosion of the gastrointestinal tract are known (Geisinger et al.(1990), Mod-Pathol., 3(5):619-624; Carlborg et al. (1983), Laryngoscope,93(2):184-187; Carlborg et al. (1980), Eur-Surg-Res., 12(4):270-282;Keshavarzian et al. (1991), Alcohol-Clin-Exp-Res., 15(1):116-121; Katzet al. (1988), Dig-Dis-Sci., 33(2):217-224; and Zeeh et al. (1996),Gastroenterology, 110(4):1077-1083, the disclosures of which are herebyincorporated by reference). Standard in vivo models of inflammatorybowel disease are well known (Morris et al. (1989), Gastroenterology,96:795-803; Rachmilewitz et al. (1989), Gastroenterology, 97:326-327;Allgayer et al. (1989), Gastroenterology, 96:1290-1300; and Kim andBorstad (1992), Scand. J. Gastroenterol, 27(7):529-537, the disclosuresof which are hereby incorporated by reference).

Animal studies have established the relationship between totalparenteral nutrition (TPN) and intestinal mucosal atrophy (Buchman etal. (1995), Journal of Parenteral and Enteral Nutrition, 19:453-460).The decrease in intestinal villus height is attributed to the lack ofgrowth stimulus provided through oral intake of nutrients. This isreflected in a reduction in the labeling index, a measure of growth.Decreases in villus height are also correlated with decreases inspecific activities of enzymes involved in nutrient absorption. KGF-2protein product(s) are useful to either protect against atrophy duringthe fasting and/or facilitate regrowth upon reintroduction of oralnutrients.

Hyaline membrane disease of premature infants results in the absence ofsurfactant production by type II pneumocytes within the lung, resultingin the collapse of the alveoli. KGF-2 protein product(s) are useful totreat and/or prevent hyaline membrane disease.

Smoke inhalation is a significant cause of morbidity and mortality inthe week following a burn injury, due to necrosis of the bronchiolarepithelium and the alveoli. KGF-2 protein products are useful treatand/or prevent inhalation injuries.

Emphysema results from the progressive loss of alveoli. KGF-2 proteinproduct(s) are useful to treat and/or prevent emphysema.

Disorders of the pancreas may be endocrine-related such as Type I orType II diabetes, or may be exocrine-related such as pancreatitis andpancreatic inefficiencies or cystic fibrosis. Patients with diagnosedType I diabetes require constant exogenous insulin administration.Patients with diagnosed Type II diabetes progress through varying stagesof insulin resistance/insufficiency to ultimately also require exogenousinsulin administration. KGF-2 protein product(s) are useful toameliorate, delay and/or circumvent permanent manifestation of diabetesmellitus or as an adjunct in the setting of islet cell transplantationby inducing pancreatic beta cell function in order to normalize bloodglucose levels during varying metabolic demands, yet avoid frequent orprofound hypoglycemia. Standard models of diabetes are known (Junod etal. (1967), Proc. Soc. Exp. Bio. Med. 126(1):201-205; Rerup (1970),Pharm. Rev., 22:485-518; Rossini et al. (1977), P.N.A.S., 74:2485-2489;and Ar'Rajab and Ahren (1993), Pancreas, 8:50-57, the disclosures ofwhich are hereby incorporated by reference). A standard model ofpancreatic cell proliferation is known (Yi et al. (1994), AmericanJournal of Pathology, 145(1):80-85, the disclosure of which is herebyincorporated by reference).

Corneal cells may be damaged by corneal abrasion and/or cornealulcerations due to chemicals, bacteria or viruses. KGF-2 proteinproduct(s) are useful treat and/or prevent corneal degeneration.Standard in vivo models of corneal cell regeneration are known (Inatomiet al. (1994), Investigative Opthalmology and Visual Science,35(4):1318, Abstract 299; Sotozono et al. (1994), InvestigativeOpthalmology and Visual Science, 35(4):1941, Abstract 317; Wilson et al.(1994), Investigative Opthalmology and Visual Science, 35(4):1319,Abstract 301; Wilson et al. (1993), The FASEB Journal, 7(3):A493,Abstract 2857; Inatomi et al. (1994), Investigative Ophthalmology &Visual Science, 35(4):1318; Wilson et al. (1994), Experimental EyeResearch, 59(6):665-678; and Sotozono et al. (1995), InvestigativeOphthalmology & Visual Science, 36(8):1524-1529, the disclosures ofwhich are hereby incorporated by reference).

KGF-2 protein product(s) are useful to treat and/or prevent gum disease.Standard in vivo models of gum disease are known.

KGF-2 protein product(s) are useful to treat and/or prevent ulceratingand/or inflammatory conditions including conditions related tochemotherapy (as discussed above) and/or infection. Standard in vivomodels of urinary bladder damage are known (Ford and Hess (1976), Arch.Intern. Med., 136:616-619 and Droller, et al. (1982), Urol., 20:256-258,the disclosures of which are hereby incorporated by reference).

KGF-2 protein product(s) are useful to treat and/or prevent eardrumdamage. Standard in vivo models of tympanic membrane perforations areknown (Clymer et al. (1996), Laryngoscope (USA), 106(3):280-285, thedisclosure of which is hereby incorporated by reference).

KGF-2 protein product(s) are useful to treat and/or prevent disorders ordamage to salivary gland tissue, including radiation/chemotherapyeffects (as discussed above) and autoimmune diseases such as Sjogren'sSyndrome which can cause salivary glandinsufficiency (sicca syndrome).Standard in vivo models of salivary gland tissue damage are known.

The following examples are included to more fully illustrate the presentinvention. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

EXAMPLES

Standard methods for many of the procedures described in the followingexamples, or suitable alternative procedures, are provided in widelyrecognized manuals of molecular biology such as, for example, Sambrooket al. (1989), supra and Ausubel et al. (1990), supra. All chemicals areeither analytical grade or USP grade.

Example 1 Protein Production

The following example teaches the production of the following KGF-2protein(s): dN29 hFGF10, dN20 hFGF10, hFGF10 and hFGF10 R149Q.

Please note that the numbering of the “dN” designation is based on thenumber of amino acids deleted from the normal N-terminal of the ratfull-length sequence. Human FGF10 has fewer amino acids at theN-terminal than rat FGF10. The number does not include the methionineadded by E. coli expression. The amino acid sequence of dN37 rFGF10 isidentical to dN29 hFGF10. The amino acid sequence of dN28 rFGF10 isidentical to dN20 hFGF10.

A. Preparation of DNA

pAMG21 dN29 rFGF10:

The plasmid pAMG21 dN29 hFGF10 contains DNA encoding the amino acidsequence set forth in FIG. 2 (dN29 hFGF10). The plasmid pAMG21 dN29hFGF10 contains a truncation of the DNA encoding the 37 amino-terminalresidues from the mature rFGF10 sequence, with the truncation having thefollowing N-terminal amino acid sequence: MSYNHLQ . . . (beginning atresidue #76, FIG. 2, Yamasaki et al., (1996), supra). Thus, dN29 hFGF10has the sequence of Ser⁶⁹ to Ser²⁰⁸ of SEQ ID NO:2 (ΔN32 KGF-2). pAMG21dN29 hFGF10 was created as follows.

First, plasmid pAMG21 dN6 rFGF10 was constructed. For this construction,PCR was performed using mature rat FGF10 cDNA (Yamasaki et al. (1996),J. Biol. Chem., 271(27):15918-15921, rFGF) in the vector pGEM-T(Promega, Madison, Wis.), termed pGEM-T rFGF10, as a template with thefollowing 5′ oligonucleotide primer (OLIGO#1), which incorporates anNdeI site, and 3′ oligonucleotide primer (OLIGO#2) which incorporates aBamHI site:

OLIGO#1: (SEQ ID NO:42) 5′-AAA CAA CAT ATG GTT TCT CCG GAG GCT ACC AACTCC-3′

OLIGO#2: (SEQ ID NO:43) 5′-AAA CAA GGA TCC TTT ATG AGT GGA CCA CCA TGGGG-3′

The PCR product generated in this reaction was purified and digestedwith restriction endonucleases NdeI and BamHI. The 525 base pair (bp)restriction-digested PCR product was purified from an agarose gel andligated with a similarly purified 6 Kilobase (Kb) BamHI to NdeI pAMG21vector DNA fragment. [The expression vector pAMG21 (ATCC accession no.98113) contains appropriate restriction sites for insertion of genesdownstream from a luxPR promoter (see U.S. Pat. No. 5,169,318 fordescription of the lux expression system).] The resultant encoded rFGF10protein differs from rFGF10 by deletion of the first 6 amino-terminalamino acid residues to a naturally occurring methionine residue, withthe protein having the following amino-terminal (N-terminal) amino acidsequence: MVSPEAT . . . (beginning at residue #43, FIG. 2, Yamasaki etal. (1996), supra).

Next, the plasmid pAMG21 His rFGF10 was constructed using a pAMG21 Hisvector. The pAMG21 His vector differs from pAMG21 as follows: betweenthe initiating methionine codon of pAMG21 (ATG) and the sequence thatfollows it (GTTAACG . . . ), the following sequence is inserted “AAA CATCAT CAC CAT CAC CAT CAT GCT AGC” which codes for “KHHHHHHHAS”. Theaddition of the codons for Ala and Ser after the 7×His tag afford aconvenient restriction site, NheI, for cloning.

The 4.7 Kb BstXI-NheI fragment of pAMG21 His plasmid vector was thenligated with the 1.8 Kb BspEI-BstXI fragment of pAMG21 dN6 rFGF10 andthe following oligonucleotide linkers OLIGO#3 and OLIGO#4 (NheI toBspEI).

OLIGO#3: (SEQ ID NO:44) 5′-CTA GCG ATG ACG ATG ATA AAC AGG CTC TGG GTCAGG ACA TGG TTT CT-3′

OLIGO#4: (SEQ ID NO:45) 5′-CCG GAG AAA CCA TGT CCT GAC CCA GAG CCT GTTTAT CAT CGT CAT CG-3′

The resultant encoded protein differs from dN6 rFGF10 by having ahistidine tag (7×His) followed by an enterokinase cleavage site with thefull-length (mature) N-terminal sequence (beginning at residue #37, FIG.1, Yamasaki et al. (1996), supra). Twenty-two amino acids were added tothe amino-terminus of the dN6 rFGF10 as follows:MKHHHHHHHASDDDDKQALGQD[MVSPEAT . . . ].

pAMG21 His rFGF10 was used as a template for PCR amplification using thefollowing 5′ oligonucleotide primer (OLIGO#5) which incorporates an NdeIsite, and 3′ oligonucleotide primer (OLIGO#6) which incorporates a BamHIsite:

OLIGO#5: (SEQ ID NO:46) 5′-GGA GGA ATA ACA TAT GTC CTA CAA TCA CCT GCAGGG AGA TGT CCG-3′

OLIGO#6: (SEQ ID NO:47) 5′-AAA CAA GGA TCC TTT ATG AGT GGA CCA CCA TGGGG-3′

The PCR product generated in this reaction was purified and then used asa template for subsequent PCR amplification with the following 5′oligonucleotide primer (OLIGO#7) which incorporates an XbaI site, and 3′oligonucleotide primer (OLIGO#8) which incorporates a BamHI site:

OLIGO#7: (SEQ ID NO:48) 5′-TTA GAT TCT AGA TTT GTT TTA ACT AAT TAA AGGAGG AAT AAC ATA TG-3′

OLIGO#8: (SEQ ID NO:49) 5′-AAA CAA GGA TCC TTT ATG AGT GGA CCA CCA TGGGG-3′

The PCR product generated in this reaction was purified and digestedwith restriction endonucleases BamHI and XbaI. The 465 bprestriction-digested product was purified from an agarose gel andligated with a similarly purified 6 Kb pAMG21 BamHI-XbaI DNA fragment toform pAMG21 dN29 hFGF10.

E. coli host strain GM120 (ATCC accession no.55764) has the lacIQpromoter and lacI gene integrated into a second site in the hostchromosome of a prototrophic E. coli K12 host. Transformation of GM120E. coli host with this ligation mixture and plating on Luria agar platescontaining 40 μg/ml kanamycin yielded recombinant bacterial colonies. Abacterial clone containing the correct recombinant plasmid wasidentified by PCR screening. Plasmid DNA was purified and sequenced toconfirm the insert sequence. Growth of recombinant bacterial cultures toexpress the gene product is described below.

pAMG21 dN20 hFGF10:

The plasmid pAMG21 dN20 hFGF10 contains DNA encoding the amino acidsequence set forth in FIG. 3 (dN20hFGF10). The plasmid pAMG21 dN20hFGF10 contains a deletion of the DNA encoding the first 28 amino acidsof the mature rFGF10 sequence resulting in the following N-terminalamino acid sequence: MSSPSSA . . . (beginning at residue #65. FIG. 2,Yamasaki et al. (1996), supra). plasmid. Thus dN20 hFGF10 has thesequence of Ser⁵⁸ to Ser²⁰⁸ of SEQ ID NO:2 (ΔN21 KGF-2).

pAMG21 dN20 hFGF10 was constructed as follows.

The 6 Kb BamHI-NdeI pAMG21 vector fragment was ligated to an NdeI-BamHIdN20 hFGF10 PCR product generated as follows: PCR was carried out usingpGEM-T rFGF10 as the template and the following 5′ oligonucleotideprimer (OLIGO#9) which incorporates an NdeI site at the 5′ end of therFGF10 gene and deletes codons for the first 28 amino acids, and 3′oligonucleotide primer (OLIGO#10) which incorporates a BamHI site at the3′ end of the rFGF10 gene:

OLIGO#9: (SEQ ID NO:50) 5′-AAA CAA CAT ATG TCT TCT CCT TCC TCT GCA GGTAGG CAT GTG CGG AGC TAC AA-3′

OLIGO#10: (SEQ ID NO:51) 5′-AAA CAA GGA TCC TTT ATG AGT GGA CCA CCA TGGGG-3′

This PCR product was purified, digested with restriction endonucleasesNdeI and BamHI and, as described above, ligated to the 6 Kb BamHI-NdeIpAMG21 vector fragment.

Transformation of GM120 E. coli host with this pAMG21 dN20 hFGF10ligation product and plating on Luria agar plates containing 40 μg/mlkanamycin yielded recombinant bacterial colonies. A bacterial clonecontaining the correct recombinant plasmid was identified by PCRscreening. Plasmid DNA was purified and sequenced to confirm the insertsequence. Growth of recombinant bacterial cultures to express the geneproduct is described below. pAMG21 hFGF10 R149Q:

The plasmid pAMG21 hFGF10 R149Q replaces an arginine residue at position149 in hFGF10 (Leu⁴⁰ to Ser²⁰⁸ of SEQ ID NO:2) with a glutamine residue(FIG. 4). pAMG21 hFGF10 R149Q was constructed as follows.

The plasmid pAMG21 rFGF10 was created by ligation of the 6.5 KbBspEI-NdeI fragment of pAMG21 His rFGF10 with the followingoligonucleotide linkers OLIGO#11 and OLIGO#12 (NdeI to BspEI).

OLIGO#11: (SEQ ID NO:52) 5′-TAT GCT GGG TCA GGA CAT GGT TTC T-3′

OLIGO#12: (SEQ ID NO:53) 5′-CCG GAG AAA CCA TGT CCT GAC CCA GCA-3′

The resultant encoded protein differs from His rFGF10 by deletion of the7×Histidine tag and restoration of the original mature amino terminalprotein sequence (MLGQDM . . . ).

A 4.8 Kb BstXI-BspEI fragment of pAMG21 rFGF10 was ligated with the 1.8Kb fragment of pAMG21 dN20 hFGF10 PstI (introduced)-BstXI and thefollowing OLIGO#13 and OLIGO#14 oligonucleotide linkers (PstI to BspEI)to delete eight serine codons from the rat sequence.

OLIGO#13: (SEQ ID NO:54) 5′-CCG GAG GCT ACC AAC TCT AGC TCC AGC AGC TTCTCC TCT CCT AGC TCT GCA-3′

OLIGO#14: (SEQ ID NO:55) 5′-GAG CTA GGA GAG GAG AAG CTG CTG GAG CTA GAGTTG GTA GCC T-3′

pAMG21 hFGF10 R149Q was constructed by the ligation of the 6.1 Kb pAMG21hFGF10 BamHI-PstI fragment with a hFGF10 R149Q PstI-BamHI PCR product.This PCR product was created as follows:

PCR A was performed using pAMG21 dN29 hFGF10 as the template with thefollowing 5′ oligonucleotide primer (OLIGO#15) and 3′ oligonucleotideprimer (OLIGO#16), which introduces a codon change AGA→CAG:

OLIGO#15: (SEQ ID NO:56) 5′-AAC ACC TAT GCA TCT TTT AAC TGG C-3′

OLIGO#16: (SEQ ID NO:57) 5′-GTC CCT GCC TGG GAG CTC CTT TTC CAT TC-3′

PCR B was performed using pAMG21 dN29 hFGF10 as the template with thefollowing 5′ oligonucleotide primer (OLIGO#17), which introduces a codonchange, and 3′ oligonucleotide primer (OLIGO#18), which incorporates aBamHI site:

OLIGO#17: (SEQ ID NO:58) 5′-GCT CCC AGG CAG GGA CAA AAA ACA AGA AGG-3′

OLIGO#18: (SEQ ID NO:59) 5′-AAC AAA GGA TCC TTT ATG AGT GGA CCA CC-3′

The products of PCR amplifications A and B above were purified andsubsequent PCR was performed using them as template with the following5′ oligonucleotide primer OLIGO#19, and OLIGO#20, which incorporates aBamHI site.

OLIGO#19: (SEQ ID NO:60) 5′-AAC ACC TAT GCA TCT TTT AAC TGG C-3′

OLIGO#20: (SEQ ID NO:61) 5′-AAC AAA GGA TCC TTT ATG AGT GGA CCA CC-3′

The product of that reaction was also purified and subsequent PCR wasperformed using it as template with the following 5′ oligonucleotideprimer OLIGO#21, which incorporates a BamHI site, and OLIGO#22:

OLIGO#21: (SEQ ID NO:62) 5′-AAC AAA GGA TCC TTT ATG AGT GGA CCA CC-3′

OLIGO#22: (SEQ ID NO:63) 5′-CCG GAG GCT ACC AAC TCT AGC TCC AGC AGC TTCTCC TCT CCT AGC TCT GCA-3′

That final PCR product was purified and digested with restrictionendonucleases PstI and BamHI. Following restriction digestion, the 440bp DNA fragment was gel purified and ligated as described above.

Transformation of GM120 E. coli host with this ligation and plating onLuria agar plates containing 40 μg/ml kanamycin yielded recombinantbacterial colonies. A bacterial clone containing the correct recombinantplasmid was identified by PCR screening. Plasmid DNA was purified andsequenced to confirm the insert sequence. Growth of recombinantbacterial cultures to express the gene product is described below.

B. Production in E. coli:

Cultures of recombinant GM120 E. coli cells containing the DNAsequence-confirmed plasmid of interest (pAMG21 dN29 rFGF10 and pAMG21hFGF10 R149Q, respectively) are each grown to optimize expression of theintroduced gene, as follows:

Five hundred milliliter flasks of Luria Broth plus Kanamycin were seededwith cells and grown at 30° C. degrees from 10 to 16 hours. All 500 mLwas added to a 9 L to 11 L of NZ amine-based media in a 15 L fermentor.All batches were grown at a pH of 7 and a dissolved oxygen levelof >50%. Batches of cells containing pAMG21 dN29 rFGF10 were grown andinduced at 37° C. and of cells containing pAMG21 hFGF10 R149Q were grownand induced at 30° C. The batches were grown at a pH of 7 and adissolved oxygen level of >50%. When optical cell density reached 10±2,autoinducer was added to the fermentor and cells were allowed to growfor 12 hours. After 12 hours, the broth was chilled to less than 15° C.,the fermentor was drained and the cells were collected bycentrifugation. The cell paste was frozen.

C. Purification

dN29 hFGF10:

dN29 hFGF10 was purified using three chromatography steps: S-Sepharoseat pH 7.5, Heparin-Sepharose at pH 7.5, and hydroxyapatite. One hundredgrams E. coli cell paste containing dN29 hFGF10 was homogenized anddisrupted exactly as described above. Following centrifugation at15,300×g for 3 hours, the supernatant-containing soluble dN29 hFGF10 wasadjusted to 40 mM Tris-HCl, pH 7.5, by addition of 1 M Tris-HCl, pH 7.5,then applied to a 300 mL S-Sepharose FF column equilibrated in 40 mMTris-HCl, pH 7.5. After washing the column with equilibration buffer toremove unbound protein, the column was eluted with a 40-volume gradientfrom 0 to 2 M NaCl in 40 mM Tris-HCl, pH 7.5. Fractions eluting between0.9 M and 1.1 M NaCl contained dN29 hFGF10, which was detected as a 16kDa band on SDS-PAGE. The identity of this band was confirmed byN-terminal sequencing. These fractions were pooled, diluted with 40 mMTris-HCl, pH 7.5, to reduce the NaCl concentration to 0.4 M, and appliedto a 60 mL Heparin-Sepharose column equilibrated in 40 mM Tris-HCl, pH7.5. The column was washed with equilibration buffer to remove unboundprotein, then eluted with an 80-volume gradient from 0 to 3 M NaCl inthe same buffer. dN29 hFGF10 eluted between 1.0 M and 1.35 M NaCl. Thesefractions were pooled and dialyzed against 40 mM Tris-HCl, pH 7.5. Thedialyzed sample was applied to a 50 mL hydroxyapatite columnequilibrated in 40 mM Tris-HCl, pH 7.5. Following sample application thecolumn was washed with equilibration buffer to remove unbound protein,then eluted with a 40-volume gradient from 0 to 0.5 M NaCl. Fractionseluting between 0.24 M and 0.44 M NaCl contained dN29 hFGF10. Thesefractions were pooled, concentrated, and buffer-exchanged to PBS. Samplepurity was estimated to be greater than 97% by Coomassie Blue-stainedSDS-gels. The yield of purified dN29 hFGF10 was 90 mg from 100 g cellpaste.

hFGF10 R149Q:

hFGF10 R149Q was purified exactly as described above for dN29 hFGF10.Human R149Q FGF10 had a lower binding affinity for Heparin-Sepharosethan dN29 hFGF10, eluting between 0.5-0.8 M NaCl. The identity andpurity of hFGF10 R149Q was analyzed by N-terminal sequencing, andSDS-gel electrophoresis.

Example 2 In Vitro Bioassay

The bioactivity of purified dN29 hFGF10 was assessed by the Balb/MKmouse keratinocyte proliferation assay, which is designed to measurespecific activity.

For the Balb/MK keratinocyte assay, 0.5 mg/mL stock solutions of dN29hFGF10 in PBS was prepared. These samples were serially diluted intoassay medium and 50 mL of each dilution was added to tissue culturewells containing Balb/MK cells in 180 mL assay medium. The finalconcentration of dN29 hFGF10 ranged from 1.2×10⁻¹ ng/mL to 2.2×10⁴ng/mL. Cell proliferation was measured by uptake of tritiated thymidine.

The estimated ED₅₀O value for dN29 hFGF10 was 46 ng/mL. The results showthat dN29 hFGF10 is effective in this assay.

Example 3 Exploratory Studies in Normal Mice

In the first study, 18 female BDF1 mice were divided into 6 groups of 3mice each (1 treated and 1 control group at each of 3 time points). Thefirst two groups received 5 mg/kg dN29 hFGF10 or the buffer control IVfor 1 day, the second two groups received 5 mg/kg dN29 hFGF10 or thebuffer control IV daily for 3 days, and the third two groups received 5mg/kg dN29 hFGF10 or the buffer control IV daily for 7 days. All micewere injected with 50 mg/kg BrdU one hour prior to harvest,radiographed, and sacrificed. Body and selected organ weights (includingall segments of small intestine) were taken, blood was drawn forhematology and serum chemistries, and organs were harvested forhistologic analysis and BrdU labeling. There was some elevation ofstomach on day 7, liver on day 3, jejenum on day 7. There was no effecton thymus. The serum chemistries were variable and normalize veryrapidly.

Example 4 Chemotherary-induced Pulmonary Fibrosis

Male Lewis rats weighing approximately 225 grams received an i.v.injection or intratracheal instillation of 5 mg/kg of dN29 hFGF10 orvehicle 72 and 48 hours prior to receiving 2.5U of bleomycin via theintratracheal route. Rat weight was monitored over the course of thefollowing 15 days at which time pulmonary function tests were performedin the rats that had received the dN29 hFGF10 via the intratrachealroute. For histology, catheters was placed in the trachea of each ratand the lungs were filled with 3 ml of formalin via hydrostaticpressure. Following fixation for 48 hours and the lungs were processedinto paraffin for sectioning and staining.

Rats receiving saline administration lost 42% of their body weight whilethose treated with dN29 hFGF10 were are 129% of their weight compared tothe day of bleomycin administration. One rat in the saline group diedbefore the time of sacrifice, this death is assumed to be from theinsult of bleomycin to the lung. There was a significant difference inpulmonary respiratory rate and tidal volume between the two groups. Ratstreated with saline had a respiration rate of 286 breaths per minutecompared with 247 for the dN29 hFGF10 group. Untreated control rats hadrespiratory rate of 216 breaths per minute which is significant vs. thedN29 hFGF10 group at the p<0.05 level.

Histologically, there was gross and microscopic changes to the salinetreated group. The lungs in this group were deformed, as was observed atthe time of sacrifice, and had excessive inflammation and fibrosis.Lungs of the dN29 hFGF10 group were very similar to those of theuntreated controls with the exception of having focal mild microscopicinflammation. There was no distinguishable gross difference between thedN29 hFGF10 and normal rats.

Example 5 Radiation-induced Mucositis Model

Mucositis is induced in mice with 12 Gy of whole-body radiation. Miceare treated daily with 5 mg/kg/day of recombinant human KGF-2 (preparedgenerally in accordance with the teachings of WO 96/25422, rhuKGF-2)beginning on the day before radiation and continuing to day threepost-radiation. Four days after radiation, the mice are necropsied andthe number of proliferating crypts (containing BUdR-positive cells) arecounted.

rhuKGF-2 treatment increases the number of proliferating crypts in theduodenum, proximal and distal jejunum of the small intestine relative tonon-rhuKGF-2 treated animals. rhuKGF-2 is also able to decrease the bodyweight loss in the irradiated mice.

Example 6 Adriamycin-induced Mucositis Model

Mucositis is induced in mice with a single intraperitoneal dose ofAdriamycin at 24 mg/kg. Mice are treated daily with 1 mg/kg/day ofrhuKGF-2 beginning on the day before radiation and continuing to daythree post-radiation. Four days after radiation, the mice are necropsiedand the number of proliferating crypts (containing BUdR-positive cells)are counted.

rhuKGF-2 treatment increases the number of proliferating crypts in theduodenum, jejunum and ileum relative to non-rhuKGF-2 treated animals.

Example 7 5-Fluorouracil-induced Mucositis Model

Mice are injected with 5-fluorouracil (5-FU, 50 mg/kg/day×4 days), aregimen that in non-treated animals leads to a survival ranging onlybetween 20-50%. rhuKGF-2 pretreatments (5 mg/kg/day×3 days), but notpost-treatments, increase survival relative to non-rhuKGF-2 treatedanimals and improvements in survival are seen at doses as low as 0.5mg/kg/day. Hepatic abscesses are commonly found in control, but notrhuKGF-2-pretreated, surviving mice indicating that 5-FU's toxicity isin part due to loss of the GI barrier function. In addition,rhuKGF-2-pretreated mice lose less weight and consume more food andwater during the 5-FU treatment period. rhuKGF-2 pretreatments alsoameliorate weight loss in mice following carboplatin (125 mg/kg×1 day)exposure, excluding the possibility that rhuKGF's effects are 5-FUspecific. rhuKGF-2 pretreatments also improve survival and weight lossnadirs in chemotherapy/radiation combination experiments when mice areinjected with a single dose of 150 or 300 mg/kg of methotrexate followedby irradiation (6 Gy) 1 hour later.

Example 8 Colitis Model

In two groups of 10 animals each, colitis is induced by colonicinstillation of 2,4,6-trinitrobenzenesulfonic acid in ethanol at a doseof 50 mg/kg body weight. To determine if rhuKGF-2 acts through aprotective mechanism, one group of rats (group A) is pretreated withrhuKGF-2 or vehicle at 24 hours and at 1 hour prior to induction ofcolitis at a dose of 5 mg/kg (i.p.) and the animals are sacrificed 8hours after injury. To assess potential healing effects, rhuKGF-2 orvehicle (same dosage, i.p.) is injected in a second group (group B) 24hours after induction of colitis and treatment is continued daily for 1week. Tissue damage is examined microscopically and is expressed aspercentage of ulcerations or erosions.

Animals which are treated with rhuKGF-2 after induction of colitis(group B) show significantly less ulcerations compared to the controlgroup (group A). In animals treated prior to induction of colitis, thereare erosions, but no ulcers are seen due to the short study period of 8hours, and the erosions are not significantly different from those seenin the control group (group A).

Example 9 Dextran Sulfate-induced Colitis Model

Study 1: Rats are fed 4% and 6% DSS in water for 1 week. At the end ofthe second week the distal 4 cm of the colon is preserved. Eightsections at 0.5 cm intervals are prepared and stained with H & E. Thepercent of each colon section with necrosis (destruction of theglandular structure) is assessed in a randomized and coded fashion.

Study 2: Rats are given IP vehicle or rhuKGF-2 (1 mg/kg/day) and fedeither water or 4% dextran sulfate sodium for 14 days. The colonicsections are stained with PAS.

Dextran sulfate sodium appears to induce a dose-related increase incolonic mucosal necrosis. rhuKGF-2 administered at 1 mg/kg/day for 14days increases colonic mucin production in the control group as well asin the dextran sulfate sodium-treated rats.

Example 10 Rat Cirrhosis Model

Male Sprague-Dawley rats weighing between 150 and 175 gms are used.Animals are exposed to phenobarbitol (0.35 mg/ml) in their drinkingwater for the duration of the study. Animals are dosed weekly with CC14in a corn oil vehicle while under light isoflurance anesthesia. Theinitial dose of CC14 is 40 μl per rat. The dose is adjusted weekly, upor down in 40 μl increments based on weight gain. Ten control animalsare exposed to phenobarbitol in their drinking water and lavaged weeklywith corn oil vehicle. Liver function is assessed by measurement ofbromosulphopthylein (BSP) clearance, serum transaminase levels and serumalbumin levels. At the time of sacrifice, livers are removed, weighedand processed for determination of hydroxyproline levels as an indicatorof collagen deposition and fibrosis.

Animals are maintained on the above cirrhosis induction protocol for 11weeks. In the eleventh week, animals are randomized into control andrhuKGF-2 treatment groups. rhuKGF-2 is given once per day bysubcutaneous injection at a dose of 1 mg/kg, for a total of 15 days.After 15 days of rhuKGF-2 treatment, the animals are euthanized.

Rats in which cirrhosis is induced with CC14 show an elevation in serumBSP concentration, reflecting impaired liver clearance of this agent.Rats treated with rhuKGF-2 have a lower BSP serum level than untreatedanimals, suggesting an improved liver function. Rats made cirrhotic byCC14 show an elevation in SGPT which is reversed by rhuKGF-2 treatment.rhuKGF-2 treatment is able to elevate serum albumin. rhuKGF-2 treatmentresults in an increased liver-to-body weight ratio, reflectingcompensatory liver growth.

Example 11 Hepatectomy Model

Rats subjected to a 70% partial hepatectomy recover their original livermass more quickly when treated with 1 mg/kg/d rhuKGF-2 than whencompared to untreated animals.

Example 12 Acute Hepatotoxicity Models

In acute hepatotoxicity models, rhuKGF-2 treatment (1 mg/kg either priorto or 3 hr after the inciting agent) blunts increases in serumtransaminase levels in rats with acute hepatic failure induced witheither carbon tetrachloride, acetaminophen or galactosamine.Pretreatment with rhuKGF-2 also prevents a decrease in liver clearancefunctions after acetaminophen, as measured by sulfobromophthalein (BSP)clearance.

Example 13 In Vivo Retro-viral-mediated Gene Transfer Model

Mice are intraveneously administered with rhuKGF-2 (1-5 mg/kg). 48 hoursafter intravenous injection of rhuKGF-2, murine hepatocyte proliferationincreases, compared to non-stimulated livers, and returns to normalproliferative levels. No modules or microscopic abnormalities are notedeither acutely or after 5 months.

When rhuKGF-2 treatment is followed by intravenous injection of hightiter E. coli LacZ expressing Moloney retroviral vectors (1×108 cfu.ml),β-galactosidase expression increases with a percentage of thehepatocytes being transduced. Several months later, a portion of thetransduced hepatocytes remain X-gal positive.

Example 14 In Vivo Model of Diabetes

Male rats weighing 200 to 260 grams at study initiation are used in thismodel (WO 9611950). Diabetes is induced by a single intravenousinjection of streptozotocin at 50 mg of streptozotocin in sodium citratebuffer per kg of body weight. Non-diabetic control rats receive a singleintravenous injection of sodium citrate buffer for control purposes.rhuKGF-2 is administered daily as a subcutaneous injection. The rhuKGF-2dose is 3 or 5 mg/kg/day, depending upon the experiment.

In the first experiment, rhuKGF-2 therapy is initiated two days beforediabetes, is induced and continued after the induction of diabetes for atotal of eight injections. Those diabetic rats which are treated withrhuKGF-2 prior to diabetes induction, and for which rhuKGF-2 is alsocontinued after the induction, show symptoms indicative of a milder formof diabetes. Thus, rhuKGF-2 therapy either partially prevents inductionof the disease or restores insulin-producing islet cells afterstreptozotocin-induced beta cell destruction.

In the second and third experiments, rhuKGF-2 therapy administeredsubcutaneously is initiated one day after the induction of diabetes withstreptozotocin. In the second study, fasting water intake and urineoutput are significantly less in the rhuKGF-2-treated diabetic rats whencompared to diabetic rats on day 9, which is further indicative ofamelioration of the disease condition. In the third study, rhuKGF-2therapy is able to increase the total content of insulin and C-peptidein the pancreas of diabetic rats when compared to diabetic rats treatedwith sodium chloride solution.

In the fourth experiment, a 7-day course of rhuKGF-2 therapy isinitiated 7 days after streptozotocin treatment and the animals are thenfollowed for an additional 12 weeks. In all experiments, except for thefourth experiment, blood glucose levels, urine glucose levels and urinevolume are used as end points for analysis. Additionally, water intake,urine C-peptide levels, or total pancreatic insulin and C-peptidecontent are measured in some experiments. In the fourth experiment, theonly assessed endpoint is blood glucose.

Because a large fraction of insulin is removed from the circulation bythe liver, measurement of peripheral insulin concentrations reflectspost-hepatic metabolism events rather than insulin secretion from thepancreas. Therefore, measurements of C-peptide are often made and usedas a peripheral marker of insulin secretion. C-peptide is produced fromthe processing of pro-insulin to insulin. Insulin and C-peptide aresecreted from the beta cells in equimolar amounts, and only a smallamount of C-peptide is extracted by the liver.

STZ-treated animals from both groups receiving rhuKGF-2 have significantdeclines in blood glucose during the rhuKGF-2 dosing period.

Example 15 Hyperoxia-induced Mortality Model

To determine the effect of rhuKGF-2 administration on hyperoxia-inducedlung injury, rats are treated by intratracheal instillation and exposedto >98% oxygen for up to 120 hours. At necropsy, after 120 hours ofhyperoxia exposure, the lungs of rhuKGF-2 treated animals appear grosslynormal, with few scattered areas of puncture hemorrhage on the pleuralsurface, compared with the grossly hemorrhagic lungs of untreated ratsdying between 55 and 80 hours of hyperoxia exposure.

Histopathologically, the lungs of untreated animals demonstrate largeareas of hemorrhage and interstitial edema. The intraaveolar spacecontains red blood cells, inflammatory cells, and proteinaceous exudate.In contrast, there is no intraaveolar exudate and minimal evidence ofhemorrhage in the lungs of the animals treated with rhuKGF-2 who survivefor 120 hours in hyperoxia.

At doses of 5 and 1 mg/kg, rhuKGF-2 significantly decreaseshyperoxia-induced mortality.

Example 16 Acute Lung Injury Model

Acute permeability pulmonary edema is induced with an injection ofα-naphthylthiourea, and lung leak is assessed in an isolated perfusedlung model over 180 minutes. Leakage is confirmed with wet/dry lungweight ratios, and the alveolar fluid protein concentration is measuredafter bronchoalveolar lavage. The effect of pretreatment with rhuKGF-2(injected intratracheally 48 hours before the experiment) onα-naphthylthiourea-induced pulmonary edema is assessed(rhuKGF-2/α-naphthylthiourea group). Control groups (Control andrhuKGF-2/Control) are also studied. Histopathology is performed for eachof the four groups.

The α-naphthylthiourea produces an acute permeability pulmonary edemadetected by lung leak over the 180-minute ex vivo period of monitoringthe isolated perfused lung. Pretreatment with rhuKGF-2 significantlyattenuates these parameters which are not significantly different fromthe control group and the rhuKGF-2/control group. Histopathology showsabundant type II pneumocyte hyperplasia in the lungs of animalspretreated with rhuKGF-2, and marked pulmonary edema in animalspretreated with α-naphthylthiourea. Less edema is apparent in therhuKGF-2/α-naphthylthiourea group.

While the present invention has been described above both generally andin terms of preferred embodiments, it is understood that othervariations and modifications will occur to those skilled in the art inlight of the description above.

63 1 627 DNA Recombinant Human CDS (1)..(624) 1 atg tgg aaa tgg ata ctgaca cat tgt gcc tca gcc ttt ccc cac ctg 48 Met Trp Lys Trp Ile Leu ThrHis Cys Ala Ser Ala Phe Pro His Leu 1 5 10 15 ccc ggc tgc tgc tgc tgctgc ttt ttg ttg ctg ttc ttg gtg tct tcc 96 Pro Gly Cys Cys Cys Cys CysPhe Leu Leu Leu Phe Leu Val Ser Ser 20 25 30 gtc cct gtc acc tgc caa gccctt ggt cag gac atg gtg tca cca gag 144 Val Pro Val Thr Cys Gln Ala LeuGly Gln Asp Met Val Ser Pro Glu 35 40 45 gcc acc aac tct tct tcc tcc tccttc tcc tct cct tcc agc gcg gga 192 Ala Thr Asn Ser Ser Ser Ser Ser PheSer Ser Pro Ser Ser Ala Gly 50 55 60 agg cat gtg cgg agc tac aat cac cttcaa gga gat gtc cgc tgg aga 240 Arg His Val Arg Ser Tyr Asn His Leu GlnGly Asp Val Arg Trp Arg 65 70 75 80 aag cta ttc tct ttc acc aag tac tttctc aag att gag aag aac ggg 288 Lys Leu Phe Ser Phe Thr Lys Tyr Phe LeuLys Ile Glu Lys Asn Gly 85 90 95 aag gtc agc ggg acc aag aag gag aac tgcccg tac agc atc ctg gag 336 Lys Val Ser Gly Thr Lys Lys Glu Asn Cys ProTyr Ser Ile Leu Glu 100 105 110 ata aca tca gta gaa atc gga gtt gtt gccgtc aaa gcc att aac agc 384 Ile Thr Ser Val Glu Ile Gly Val Val Ala ValLys Ala Ile Asn Ser 115 120 125 aac tat tac tta gcc atg aac aag aag gggaaa ctc tat ggc tca aaa 432 Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly LysLeu Tyr Gly Ser Lys 130 135 140 gaa ttt aac aat gac tgt aag ctg aag gagagg ata gag gaa aat gga 480 Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu ArgIle Glu Glu Asn Gly 145 150 155 160 tac aat acc tat gca tca ttt aac tggcag cat aat ggg agg caa atg 528 Tyr Asn Thr Tyr Ala Ser Phe Asn Trp GlnHis Asn Gly Arg Gln Met 165 170 175 tat gtg gca ttg aat gga aaa gga gctcca agg aga gga cag aaa aca 576 Tyr Val Ala Leu Asn Gly Lys Gly Ala ProArg Arg Gly Gln Lys Thr 180 185 190 cga agg aaa aac acc tct gct cac tttctt cca atg gtg gta cac tca 624 Arg Arg Lys Asn Thr Ser Ala His Phe LeuPro Met Val Val His Ser 195 200 205 tag 627 2 208 PRT Recombinant Human2 Met Trp Lys Trp Ile Leu Thr His Cys Ala Ser Ala Phe Pro His Leu 1 5 1015 Pro Gly Cys Cys Cys Cys Cys Phe Leu Leu Leu Phe Leu Val Ser Ser 20 2530 Val Pro Val Thr Cys Gln Ala Leu Gly Gln Asp Met Val Ser Pro Glu 35 4045 Ala Thr Asn Ser Ser Ser Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly 50 5560 Arg His Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg 65 7075 80 Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys Ile Glu Lys Asn Gly 8590 95 Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser Ile Leu Glu100 105 110 Ile Thr Ser Val Glu Ile Gly Val Val Ala Val Lys Ala Ile AsnSer 115 120 125 Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr GlySer Lys 130 135 140 Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg Ile GluGlu Asn Gly 145 150 155 160 Tyr Asn Thr Tyr Ala Ser Phe Asn Trp Gln HisAsn Gly Arg Gln Met 165 170 175 Tyr Val Ala Leu Asn Gly Lys Gly Ala ProArg Arg Gly Gln Lys Thr 180 185 190 Arg Arg Lys Asn Thr Ser Ala His PheLeu Pro Met Val Val His Ser 195 200 205 3 426 DNA Recombinant Human CDS(1)..(423) 3 atg tcc tac aat cac ctg cag gga gat gtc cgc tgg aga aag ctgttc 48 Met Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe 15 10 15 tcc ttc acc aag tac ttt ctc aag att gaa aag aac ggc aag gtc agc96 Ser Phe Thr Lys Tyr Phe Leu Lys Ile Glu Lys Asn Gly Lys Val Ser 20 2530 ggg acc aag aag gaa aac tgt ccg tac agt atc cta gag ata aca tca 144Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser Ile Leu Glu Ile Thr Ser 35 40 45gtg gaa atc gga gtt gtt gcc gtc aaa gcc att aac agc aac tat tac 192 ValGlu Ile Gly Val Val Ala Val Lys Ala Ile Asn Ser Asn Tyr Tyr 50 55 60 ttagcc atg aac aag aag ggg aaa ctc tat ggc tca aaa gaa ttt aac 240 Leu AlaMet Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn 65 70 75 80 aatgac tgt aaa ctg aaa gag agg ata gag gaa aat gga tac aac acc 288 Asn AspCys Lys Leu Lys Glu Arg Ile Glu Glu Asn Gly Tyr Asn Thr 85 90 95 tat gcatct ttt aac tgg cag cac aac ggc agg caa atg tat gtg gca 336 Tyr Ala SerPhe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Ala 100 105 110 ttg aatgga aaa gga gct ccc agg aga gga caa aaa aca aga agg aaa 384 Leu Asn GlyLys Gly Ala Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys 115 120 125 aac acctcc gct cac ttc ctc ccc atg gtg gtc cac tca taa 426 Asn Thr Ser Ala HisPhe Leu Pro Met Val Val His Ser 130 135 140 4 141 PRT Recombinant Human4 Met Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe 1 5 1015 Ser Phe Thr Lys Tyr Phe Leu Lys Ile Glu Lys Asn Gly Lys Val Ser 20 2530 Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser Ile Leu Glu Ile Thr Ser 35 4045 Val Glu Ile Gly Val Val Ala Val Lys Ala Ile Asn Ser Asn Tyr Tyr 50 5560 Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn 65 7075 80 Asn Asp Cys Lys Leu Lys Glu Arg Ile Glu Glu Asn Gly Tyr Asn Thr 8590 95 Tyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Ala100 105 110 Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr Arg ArgLys 115 120 125 Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 130135 140 5 459 DNA Recombinant Human CDS (1)..(456) 5 atg tct tct cct tcctct gca ggt agg cat gtg cgg agc tac aat cac 48 Met Ser Ser Pro Ser SerAla Gly Arg His Val Arg Ser Tyr Asn His 1 5 10 15 ctc cag gga gat gtccgc tgg aga aag ctg ttc tcc ttc acc aag tac 96 Leu Gln Gly Asp Val ArgTrp Arg Lys Leu Phe Ser Phe Thr Lys Tyr 20 25 30 ttt ctc aag att gaa aagaac ggc aag gtc agc ggg acc aag aag gaa 144 Phe Leu Lys Ile Glu Lys AsnGly Lys Val Ser Gly Thr Lys Lys Glu 35 40 45 aac tgt ccg tac agt atc ctagag ata aca tca gtg gaa atc gga gtt 192 Asn Cys Pro Tyr Ser Ile Leu GluIle Thr Ser Val Glu Ile Gly Val 50 55 60 gtt gcc gtc aaa gcc att aac agcaac tat tac tta gcc atg aac aag 240 Val Ala Val Lys Ala Ile Asn Ser AsnTyr Tyr Leu Ala Met Asn Lys 65 70 75 80 aag ggg aaa ctc tat ggc tca aaagaa ttt aac aat gac tgt aaa ctg 288 Lys Gly Lys Leu Tyr Gly Ser Lys GluPhe Asn Asn Asp Cys Lys Leu 85 90 95 aaa gag agg ata gag gaa aat gga tacaac acc tat gca tct ttt aac 336 Lys Glu Arg Ile Glu Glu Asn Gly Tyr AsnThr Tyr Ala Ser Phe Asn 100 105 110 tgg cag cac aac ggc agg caa atg tatgtg gca ttg aat gga aaa gga 384 Trp Gln His Asn Gly Arg Gln Met Tyr ValAla Leu Asn Gly Lys Gly 115 120 125 gct ccc agg aga gga caa aaa aca agaagg aaa aac acc tcc gct cac 432 Ala Pro Arg Arg Gly Gln Lys Thr Arg ArgLys Asn Thr Ser Ala His 130 135 140 ttc ctc ccc atg gtg gtc cac tca taa459 Phe Leu Pro Met Val Val His Ser 145 150 6 152 PRT Recombinant Human6 Met Ser Ser Pro Ser Ser Ala Gly Arg His Val Arg Ser Tyr Asn His 1 5 1015 Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe Ser Phe Thr Lys Tyr 20 2530 Phe Leu Lys Ile Glu Lys Asn Gly Lys Val Ser Gly Thr Lys Lys Glu 35 4045 Asn Cys Pro Tyr Ser Ile Leu Glu Ile Thr Ser Val Glu Ile Gly Val 50 5560 Val Ala Val Lys Ala Ile Asn Ser Asn Tyr Tyr Leu Ala Met Asn Lys 65 7075 80 Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu 8590 95 Lys Glu Arg Ile Glu Glu Asn Gly Tyr Asn Thr Tyr Ala Ser Phe Asn100 105 110 Trp Gln His Asn Gly Arg Gln Met Tyr Val Ala Leu Asn Gly LysGly 115 120 125 Ala Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr SerAla His 130 135 140 Phe Leu Pro Met Val Val His Ser 145 150 7 513 DNARecombinant Human CDS (1)..(510) 7 atg ctg ggt cag gac atg gtt tct ccggag gct acc aac tct agc tcc 48 Met Leu Gly Gln Asp Met Val Ser Pro GluAla Thr Asn Ser Ser Ser 1 5 10 15 agc agc ttc tcc tct cct agc tct gcaggt agg cat gtg cgg agc tac 96 Ser Ser Phe Ser Ser Pro Ser Ser Ala GlyArg His Val Arg Ser Tyr 20 25 30 aat cac ctc cag gga gat gtc cgc tgg agaaag ctg ttc tcc ttc acc 144 Asn His Leu Gln Gly Asp Val Arg Trp Arg LysLeu Phe Ser Phe Thr 35 40 45 aag tac ttt ctc aag att gaa aag aac ggc aaggtc agc ggg acc aag 192 Lys Tyr Phe Leu Lys Ile Glu Lys Asn Gly Lys ValSer Gly Thr Lys 50 55 60 aag gaa aac tgt ccg tac agt atc cta gag ata acatca gtg gaa atc 240 Lys Glu Asn Cys Pro Tyr Ser Ile Leu Glu Ile Thr SerVal Glu Ile 65 70 75 80 gga gtt gtt gcc gtc aaa gcc att aac agc aac tattac tta gcc atg 288 Gly Val Val Ala Val Lys Ala Ile Asn Ser Asn Tyr TyrLeu Ala Met 85 90 95 aac aag aag ggg aaa ctc tat ggc tca aaa gaa ttt aacaat gac tgt 336 Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn AsnAsp Cys 100 105 110 aaa ctg aaa gag agg ata gag gaa aat gga tac aac acctat gca tct 384 Lys Leu Lys Glu Arg Ile Glu Glu Asn Gly Tyr Asn Thr TyrAla Ser 115 120 125 ttt aac tgg cag cac aac ggc agg caa atg tat gtg gcattg aat gga 432 Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Ala LeuAsn Gly 130 135 140 aaa gga gct ccc agg cag gga caa aaa aca aga agg aaaaac acc tcc 480 Lys Gly Ala Pro Arg Gln Gly Gln Lys Thr Arg Arg Lys AsnThr Ser 145 150 155 160 gct cac ttc ctc ccc atg gtg gtc cac tca taa 513Ala His Phe Leu Pro Met Val Val His Ser 165 170 8 170 PRT RecombinantHuman 8 Met Leu Gly Gln Asp Met Val Ser Pro Glu Ala Thr Asn Ser Ser Ser1 5 10 15 Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly Arg His Val Arg SerTyr 20 25 30 Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe Ser PheThr 35 40 45 Lys Tyr Phe Leu Lys Ile Glu Lys Asn Gly Lys Val Ser Gly ThrLys 50 55 60 Lys Glu Asn Cys Pro Tyr Ser Ile Leu Glu Ile Thr Ser Val GluIle 65 70 75 80 Gly Val Val Ala Val Lys Ala Ile Asn Ser Asn Tyr Tyr LeuAla Met 85 90 95 Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn AsnAsp Cys 100 105 110 Lys Leu Lys Glu Arg Ile Glu Glu Asn Gly Tyr Asn ThrTyr Ala Ser 115 120 125 Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr ValAla Leu Asn Gly 130 135 140 Lys Gly Ala Pro Arg Gln Gly Gln Lys Thr ArgArg Lys Asn Thr Ser 145 150 155 160 Ala His Phe Leu Pro Met Val Val HisSer 165 170 9 4 PRT Recombinant Human 9 Val Arg Ser Tyr 1 10 5 PRTRecombinant Human 10 His Val Arg Ser Tyr 1 5 11 6 PRT Recombinant Human11 Arg His Val Arg Ser Tyr 1 5 12 7 PRT Recombinant Human 12 Gly Arg HisVal Arg Ser Tyr 1 5 13 8 PRT Recombinant Human 13 Ala Gly Arg His ValArg Ser Tyr 1 5 14 9 PRT Recombinant Human 14 Ser Ala Gly Arg His ValArg Ser Tyr 1 5 15 10 PRT Recombinant Human 15 Ser Ser Ala Gly Arg HisVal Arg Ser Tyr 1 5 10 16 11 PRT Recombinant Human 16 Pro Ser Ser AlaGly Arg His Val Arg Ser Tyr 1 5 10 17 12 PRT Recombinant Human 17 SerPro Ser Ser Ala Gly Arg His Val Arg Ser Tyr 1 5 10 18 13 PRT RecombinantHuman 18 Ser Ser Pro Ser Ser Ala Gly Arg His Val Arg Ser Tyr 1 5 10 1914 PRT Recombinant Human 19 Phe Ser Ser Pro Ser Ser Ala Gly Arg His ValArg Ser Tyr 1 5 10 20 15 PRT Recombinant Human 20 Ser Phe Ser Ser ProSer Ser Ala Gly Arg His Val Arg Ser Tyr 1 5 10 15 21 16 PRT RecombinantHuman 21 Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly Arg His Val Arg Ser Tyr1 5 10 15 22 17 PRT Recombinant Human 22 Ser Ser Ser Phe Ser Ser Pro SerSer Ala Gly Arg His Val Arg Ser 1 5 10 15 Tyr 23 18 PRT RecombinantHuman 23 Ser Ser Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly Arg His Val Arg1 5 10 15 Ser Tyr 24 19 PRT Recombinant Human 24 Ser Ser Ser Ser Ser PheSer Ser Pro Ser Ser Ala Gly Arg His Val 1 5 10 15 Arg Ser Tyr 25 20 PRTRecombinant Human 25 Asn Ser Ser Ser Ser Ser Phe Ser Ser Pro Ser Ser AlaGly Arg His 1 5 10 15 Val Arg Ser Tyr 20 26 21 PRT Recombinant Human 26Thr Asn Ser Ser Ser Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly Arg 1 5 1015 His Val Arg Ser Tyr 20 27 22 PRT Recombinant Human 27 Ala Thr Asn SerSer Ser Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly 1 5 10 15 Arg His ValArg Ser Tyr 20 28 23 PRT Recombinant Human 28 Glu Ala Thr Asn Ser SerSer Ser Ser Phe Ser Ser Pro Ser Ser Ala 1 5 10 15 Gly Arg His Val ArgSer Tyr 20 29 24 PRT Recombinant Human 29 Pro Glu Ala Thr Asn Ser SerSer Ser Ser Phe Ser Ser Pro Ser Ser 1 5 10 15 Ala Gly Arg His Val ArgSer Tyr 20 30 25 PRT Recombinant Human 30 Ser Pro Glu Ala Thr Asn SerSer Ser Ser Ser Phe Ser Ser Pro Ser 1 5 10 15 Ser Ala Gly Arg His ValArg Ser Tyr 20 25 31 26 PRT Recombinant Human 31 Val Ser Pro Glu Ala ThrAsn Ser Ser Ser Ser Ser Phe Ser Ser Pro 1 5 10 15 Ser Ser Ala Gly ArgHis Val Arg Ser Tyr 20 25 32 27 PRT Recombinant Human 32 Met Val Ser ProGlu Ala Thr Asn Ser Ser Ser Ser Ser Phe Ser Ser 1 5 10 15 Pro Ser SerAla Gly Arg His Val Arg Ser Tyr 20 25 33 28 PRT Recombinant Human 33 AspMet Val Ser Pro Glu Ala Thr Asn Ser Ser Ser Ser Ser Phe Ser 1 5 10 15Ser Pro Ser Ser Ala Gly Arg His Val Arg Ser Tyr 20 25 34 29 PRTRecombinant Human 34 Gln Asp Met Val Ser Pro Glu Ala Thr Asn Ser Ser SerSer Ser Phe 1 5 10 15 Ser Ser Pro Ser Ser Ala Gly Arg His Val Arg SerTyr 20 25 35 30 PRT Recombinant Human 35 Gly Gln Asp Met Val Ser Pro GluAla Thr Asn Ser Ser Ser Ser Ser 1 5 10 15 Phe Ser Ser Pro Ser Ser AlaGly Arg His Val Arg Ser Tyr 20 25 30 36 31 PRT Recombinant Human 36 LeuGly Gln Asp Met Val Ser Pro Glu Ala Thr Asn Ser Ser Ser Ser 1 5 10 15Ser Phe Ser Ser Pro Ser Ser Ala Gly Arg His Val Arg Ser Tyr 20 25 30 3732 PRT Recombinant Human 37 Ala Leu Gly Gln Asp Met Val Ser Pro Glu AlaThr Asn Ser Ser Ser 1 5 10 15 Ser Ser Phe Ser Ser Pro Ser Ser Ala GlyArg His Val Arg Ser Tyr 20 25 30 38 33 PRT Recombinant Human 38 Gln AlaLeu Gly Gln Asp Met Val Ser Pro Glu Ala Thr Asn Ser Ser 1 5 10 15 SerSer Ser Phe Ser Ser Pro Ser Ser Ala Gly Arg His Val Arg Ser 20 25 30 Tyr39 34 PRT Recombinant Human 39 Cys Gln Ala Leu Gly Gln Asp Met Val SerPro Glu Ala Thr Asn Ser 1 5 10 15 Ser Ser Ser Ser Phe Ser Ser Pro SerSer Ala Gly Arg His Val Arg 20 25 30 Ser Tyr 40 4 PRT Recombinant Human40 Met Val Val His 1 41 5 PRT Recombinant Human 41 Met Val Val His Ser 15 42 36 DNA Recombinant Human 42 aaacaacata tggtttctcc ggaggctacc aactcc36 43 35 DNA Recombinant Human 43 aaacaaggat cctttatgag tggaccacca tgggg35 44 47 DNA Recombinant Human 44 ctagcgatga cgatgataaa caggctctgggtcaggacat ggtttct 47 45 47 DNA Recombinant Human 45 ccggagaaaccatgtcctga cccagagcct gtttatcatc gtcatcg 47 46 45 DNA Recombinant Human46 ggaggaataa catatgtcct acaatcacct gcagggagat gtccg 45 47 35 DNARecombinant Human 47 aaacaaggat cctttatgag tggaccacca tgggg 35 48 47 DNARecombinant Human 48 ttagattcta gatttgtttt aactaattaa aggaggaata acatatg47 49 35 DNA Recombinant Human 49 aaacaaggat cctttatgag tggaccacca tgggg35 50 53 DNA Recombinant Human 50 aaacaacata tgtcttctcc ttcctctgcaggtaggcatg tgcggagcta caa 53 51 35 DNA Recombinant Human 51 aaacaaggatcctttatgag tggaccacca tgggg 35 52 25 DNA Recombinant Human 52 tatgctgggtcaggacatgg tttct 25 53 27 DNA Recombinant Human 53 ccggagaaac catgtcctgacccagca 27 54 51 DNA Recombinant Human 54 ccggaggcta ccaactctagctccagcagc ttctcctctc ctagctctgc a 51 55 43 DNA Recombinant Human 55gagctaggag aggagaagct gctggagcta gagttggtag cct 43 56 25 DNA RecombinantHuman 56 aacacctatg catcttttaa ctggc 25 57 29 DNA Recombinant Human 57gtccctgcct gggagctcct tttccattc 29 58 30 DNA Recombinant Human 58gctcccaggc agggacaaaa aacaagaagg 30 59 29 DNA Recombinant Human 59aacaaaggat cctttatgag tggaccacc 29 60 25 DNA Recombinant Human 60aacacctatg catcttttaa ctggc 25 61 29 DNA Recombinant Human 61 aacaaaggatcctttatgag tggaccacc 29 62 29 DNA Recombinant Human 62 aacaaaggatcctttatgag tggaccacc 29 63 51 DNA Recombinant Human 63 ccggaggctaccaactctag ctccagcagc ttctcctctc ctagctctgc a 51

We claim:
 1. A keratinocyte growth factor-2 (KGF-2) protein selectedfrom the group consisting of: (a) a KGF-2 protein consisting of residues66 through 208 of the amino acid sequence set forth in SEQ ID NO: 2; and(b) a KGF-2 protein consisting of residues 66 through 208 of the aminoacid sequence set forth in SEQ ID NO: 2 and an N-terminal methionine. 2.The KGF-2 protein according to claim 1, wherein said amino acid sequenceis nonglycosylated.
 3. The KGF-2 protein according to claim 1, whereinsaid amino acid sequence is glycosylated.
 4. A chemical derivativecomprising a water-soluble polymer conjugated to the KGF-2 proteinaccording to claim
 1. 5. A polynucleotide encoding the KGF-2 proteinaccording to claim
 1. 6. A vector comprising a polynucleotide of claim 5operatively linked to an expression control sequence.
 7. A prokaryoticor eukaryotic host cell containing a polynucleotide of claim
 5. 8. Amethod comprising culturing the host cell of claim 7 in a suitablenutrient medium.
 9. The method according to claim 8, wherein said hostcell is an E. coli cell.
 10. The method according to claim 8, whereinsaid host cell is selected from a baculovirus cell, COS cell or Chinesehamster ovary cell.
 11. The method of claims 8, 9 or 10 furthercomprising isolating a keratinocyte growth factor-2 (KG F-2) proteinfrom said cultured cells or said nutrient medium.
 12. A pharmaceuticalcomposition comprising a keratinocyte growth factor-2 (KGF-2) proteinisolated in accordance with the method of claim 11 in association with apharmaceutically acceptable vehicle.
 13. A method comprising the step ofisolating a keratinocyte growth factor-2 (KGF-2) protein from a hostcell containing a polynucleotide of claim 5 cultured under conditionsallowing the expression of the KGF-2 protein by said host cell.
 14. Themethod according to claim 13 comprising the step of conjugating theisolated KGF-2 protein to a water-soluble polymer to generate a compoundcapable of stimulating the production of epithelial cells.
 15. A methodcomprising the steps of: (a) culturing a prokaryotic or eukaryotic hostcell containing a polynucleotide of claim 5; and (b) maintaining saidhost cell under conditions allowing the expression of a keratinocytegrowth factor-2 (KGF-2) protein by said host cell.
 16. The method ofclaim 15, further comprising after step (b) the following step (c): (c)isolating the KGF-2 protein expressed by said host cell.
 17. Apharmaceutical composition comprising a keratinocyte growth factor-2(KGF-2) protein isolated in accordance with the method of claim 16 inassociation with a pharmaceutically acceptable vehicle.
 18. A KGF-2protein which is the recombinant expression product of a prokaryotic oreukaryotic host cell containing an exogenous polynucleotide of claim 5.19. A pharmaceutical composition comprising the KGF-2 protein accordingto claim 1 in association with a pharmaceutically acceptable vehicle.20. The KGF-2 protein according to claim 1, wherein at least one domainof the constant region of the heavy chain of human immunoglobulin isfused to the C-terminal end of the KGF-2 protein.
 21. A recombinantfusion protein comprising the KGF-2 protein of claim 1 fused to aheterologous protein.